Biochemistry
                     Part 1 Biomolecules
                               Introduction

1 What is Biochemistry?...
•Agriculture: Herbicides and pesticides
•Medicine : Monocloning antibodies
•Nutrition : Vitamines
•Clinical Chemistry: tra...
2.3 pH
•Acids and Bases: Proton Donors and Acceptors
•pH = -log[H+]
•Weak Acid and Base Equilibria
•Ka                    ...
2.5 Entropy and the Second Law of Thermodynamics
•Entropy (S) Tendency of Systems of Molecules to Randomization
S = klnW (...
–the lichen, which consists of a fungus and an alga
   –the root nodule system formed by a leguminous plant and anaerobic
...
Chapter 1 Carbohydrates
•Carbohydrate:    a polyhydroxyaldehyde or polyhydroxyketone, or a
substance that gives these comp...
aldohexoses.
•Note that the third of these is an amino sugar
   •also shown is the most common 2-keto-D-hexose
Cyclic Stru...
group by a variety of reducing agents, including H2/M and NaBH4
   •reduction of the C=O group of a monosaccharide gives a...
α-1,6-glycosidic bonds
    •amylases catalyze hydrolysis of α-1,4-glycosidic bonds
    •debranching enzymes catalyze the h...
Glycoproteins
•Glycoproteins contain carbohydrate units covalently bonded to a
polypeptide chain
   •antibodies are glycop...
Chapter 2 Lipids and Membranes
Lipids
•Lipids: a heterogeneous class of naturally occurring organic compounds
classified t...
Mg(II), and Fe(III) ions (hard water)
                               water

Phosphoacylglycerols
•Phosphoacylglycerols (ph...
•responsible for the development of male secondary sex characteristics
Estrogens
•Estrogens: female sex hormones
   •synth...
•Integral proteins
    •bound tightly to the interior of the membrane
    •can be removed by treatment with detergents or ...
Chapter 3 Amino Acids and Peptides
Amino Acids
•Amino acid: a compound that contains both an amino group and a carboxyl
gr...
Titration of Amino Acids
Figure (a) Titration of alanine with NaOH
Figure (b) Titration of histidine with NaOH
Acidity: Aα...
•at this pH, an α-amino group is 99.9% in the acid or protonated form
    and has a charge of +1
Henderson-Hasselbalch
•We...
•Peptide  bond: the special name given to the amide bond between the -
carboxyl group of one amino acid and the c -amino g...
Chapter 4 Proteins

Protein Structure
•1° structure: the sequence of amino acids in a polypeptide chain, read from
the N-t...
•C=O---H-N     hydrogen bonds are between adjacent sheets and
   perpendicular to the direction of the sheet
β -TURN
T α-H...
structure
•Quaternary (4°) structure: the association of polypeptide chains into
aggregations

•Proteins are divided into ...
•hydrophobic interaction between nonpolar side chains, e.g., Val and Ile
   •electrostatic attraction between side chains ...
•nonetheless, in the protein-dense environment of a cell, proteins may
   begin to fold incorrectly or may associate with ...
Cooperativity of Binding/Release

The oxygenation state (filled or empty) of one site of the multisubunit
  hemoglobin can...
•Fig
Evolution of Myoglobin / Hemoglobin Proteins
•Out of the 153 amino acids in the amino acid sequences of sperm whale
m...
•3 Disulfide bonds covalently join the heavy and light chains, conferring
stability on this secreted protein (Some antibod...
•Which   is type I turn that connects β-strands at the carboxyl-terminus

Structure of Protein G(side view)
•5. hydrophili...
•7.The display is Spacefill with Structure coloring except that three strands
of the β -sheet are shown as Sticks. One str...
•
•7. Protein G is shown in backbone display with RasMol Structure coloring.
But someone has tampered with this picture! I...
Chapter 5 Neucleic Acids
•
•5.1 Two Nucleic Acids: DNA and RNA
•5.2 Propertie of nucleotides 核苷酸
•5.3 Primary Structure of...
NAD
NADP
FAD
Nucleotide propertics
1. Dissociation in water
•The phosphate group of a nucleotide acts as a strong acid (pK...
•Gene is a particular DNA sequence
 DNA is genetic substance.
Hershey and Chase - Bacteriophage T2 transfers DNA to bacter...
GGCGCGCC
CCGCGCGG

H-DNA
•purines/pyrimidines on one strand

5.5 Tertiary structure of DNA and RNA
•Tertiary structure ref...
tRNA

4.6 The Biological Functions of Nucleic Acids
•1、Genome
•2、Transcription
•3、Translation:
•rRNA(ribosome: Ribozyme, S...
Chapter 6 Enzymes : A Biological Catalyst


Important Features of Enzyme Catalysis
  1. With the exception of some RNAs th...
the change in free energy is

•The change in free energy is related to the equilibrium constant, Keq, for the
reaction by
...
–order of reaction: the sum of the exponents in the rate equation

Velocity of Enzyme Catalysis
   –consider the reaction
...
–a plot of 1/V versus 1/[S] will give a straight line with slope of KM/Vmax
   and y intercept of 1/Vmax
   –such a plot i...
•Allosteric Control - non-competitive inhibitors are allosteric inhibitors and
activators.

Competitive Inhibition
  –subs...
–these substances change the enzyme’s activity by altering the
   conformation(s) of its 4° structure
•Allosteric effector...
peptide to be cleaved often forms one or more hydrogen bonds with the
substrate:
•Trypsin cleaves after Lys and Arg residu...
•VI: Decomposition of acyl intermediate and release of the second product.
Enzyme is in the same form as in panel I!

Enzy...
–homotropic effects: allosteric interactions that occur when several
   identical molecules are bound to the protein; e.g....
Two types of allosteric enzymes
•For an allosteric enzyme, the substrate concentration at one-half Vmax is
called the K0.5...
NAD + in some dehydrogenases
• Ethanol + NAD+ <=> Acetaldehyde + NADH (Alcohol Dehydrogenase)
   NAD + in UDP-galactose ep...
Chapter 7         Hormone and Cell Signaling

The word hormone
 The word hormone is derived from the Greek verb horman, m...
 The water-soluble peptide and amine hormones do not penetrate
         cell membranes readily; their receptors are locat...
  Binding of epinephrine causes the receptor to catalyze the
       displacement of the GDP bound to inactive Gs by GTP; ...
   A third class of signal receptors and diacylglycerol and inositol-1,4,5
    -trisphosphate
      The signal receptors...
Part 2 Metabolism
         Chapter       8 Overview of Metabolism
Things to learn from this lecture
What is metabolism
T...
Metabolic pathways have committed step
Metabolic pathways are regulated
Metabolic pathways are compartmentalized ( in e...
∆Go       standard free energy change
          T = 298 K, concentration of all reactants is 1.0 M
      R = 1.98 cal/(mol...
Pathway
Energetic
Regulation
Cellular function /Location
Citric Acid Cycle
Pathway
Energetic
Regulation
Cellular f...
Transcription
•Differences between DNA and RNA polymerases
•E. coli RNA polymerase subunits and their function
•Promoters
...
Chapter 9 Energy Metabolism

9.1 glycolysis-Anaerobic Metabolism of glucose
Things to Learn
•Pathway
•Energetics
•Regulati...
–Reconstructed all the transformation steps from glycogen to lactic acid
   in vitro; revealed that many reactions of lact...
The “high energy” acyl thioester is attacked by Pi to yield the acyl phosphate
(~P) product. This step can be bypassed by ...
•a pathway is controlled at rate-limiting steps
•Flux through the rate-determining steps may be altered by several
mechani...
transported to various tissues for oxidative degradation via the glycolytic
pathway.
9 Glycogen Breakdown
•Glycogen
   –1-...
•phosphorylation promotes transition of b (less active ) state to the a(active)
state.
•The cAMP cascade activates glycoge...
more ATP in electron transport and oxidative phosphorylation
•A pathway providing many precursors for biosynthesis
citric ...
2 Acetyl CoA
  TCAcycle
     6 NADH
     2 FADH2
     2 GTP
                                        total 30-32ATP
The Fat...
•Acetate-based growth - net synthesis of carbohydrates and other
intermediates from acetate - is not possible with TCA
•Gl...
Triacylglycerol degradation
Why Fatty Acids are used for storage of energy?
Two reasons:
   –The carbon in fatty acids (mo...
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  1. 1. Biochemistry Part 1 Biomolecules Introduction 1 What is Biochemistry? •Describe structure, organization, function of cells in molecular terms. Structural Chemistry Metabolism Molecular Genetics 1.1 Biochemistry as a Discipline:Chemical Science and Biological Science (1) Biochemistry as a Chemical Science ☉Chemical Elements of Living Matter C,H,O,N,S,P,…… ☉Molecules of Living things Amino acids Sugars Lipids Nucleotides Vitamins Hormones ☉Monomers/Polymers Sugar/Polysaccharide Nucleotide/Nucleic Acids Amino acid/Polypeptides (2) Biochemistry as a Biological Science Distinguishing Characteristics of Living Matter •Constant renewal of a highly ordered structure accompanied by an increase in complexity of that structure •Overcoming entropy requires energy •Life is self-replicating 1.2 Range of sizes of objects studied by biochemists and biologists Uses of Biochemistry
  2. 2. •Agriculture: Herbicides and pesticides •Medicine : Monocloning antibodies •Nutrition : Vitamines •Clinical Chemistry: transaminases and bilirubin/Liver disease •Pharmacology: penicillin (inhibiting an enzyme that synthesizes an essential polysaccharide of the bacterial cell wall) •Toxicology: designer drugs •If the target site for action of a drug is a protein enzyme or receptor, determining the detailed molecular structure of that target allows us to design inhibitors that bind to it with great selectivity. 2.Background 2.1 Covalent and Noncovalent bond (1)Covalent Bonds (300-400 kJ/mol) Non-Covalent Interactions (2-40 kJ/mol) Hydrogen Bonds Charge-charge interactions Other non-covalent interactions (2)Charge-Charge Interactions •Coulomb's law •F = k*(q1q2)/r2 •dielectric medium, •F = k*(q1q2)/ ε r2 (3) Hydrogen Bonds •hydrogen bond donor :to which hydrogen is covalently bonded •hydrogen bond acceptor : with the nonbonded electron pair 2.2 The Role of Water in Biological Processes •Hydrophilic molecules in Aqueous Solution •Hydrophobic molecules in Aqueous Solution Clathrate cages •Amphipathic molecules in Aqueous Solution Amphipathic molecules
  3. 3. 2.3 pH •Acids and Bases: Proton Donors and Acceptors •pH = -log[H+] •Weak Acid and Base Equilibria •Ka and pKa Ka=[H+][A-]/[HA] ⇒ pKa=pH-log [A-]/[HA] •Polyprotic Acids The pH Scale and the Physiologic pH Range Henderson-Hasselbalch Equation, pI •Titration of Weak Acids: The Henderson-Hasselbalch Equation: •pH = pKa +log [A-]/[HA] •pI : Each molecule has a distinct pH (called the pI or isoelectric point) at which the net average charge of all the groups adds up to zero. If acidic groups predominate, the pI will be low. If basic groups predominate, the pI will be high. Acid dissociation of glycine •Glycine has only a single acidic group and a single basic group, the pI can be determined by averaging the pKas of the two groups (from Equation 2.18) For example, the pKa values of the carboxylate and amino groups on glycine are 2.3 and 9.6, respectively. Thus, •pI = (2.3 + 9.6)/2 = 5.95 The relative concentrations of the three forms of glycine as a function of pH. 2.4 Interactions Between Macroions in Solution •Solubility of Macroions and pH •Repulsive effects (nucleic acids) •Attractive effects (histones to DNA) •Minimum solubility at isoelectric point •Influence of Small Ions: Ionic Strength •Electrostatic interactions between macroions Debye-Huckel Theory •Salting In - adding counterions to a point increases protein solubility •Salting Out - adding very large amounts of counterions decreases protein solubility
  4. 4. 2.5 Entropy and the Second Law of Thermodynamics •Entropy (S) Tendency of Systems of Molecules to Randomization S = klnW (k = Boltzmann constant) Second Law •"The entropy of an isolated system will tend to increase to a maximum value" 2.6 Free Energy: The Second Law in Open Systems G = H - T S G <0 means Exergonic, favorable process G >0 means Endergonic, reverse process favored Free Energy and Chemical Reactons: Chemical Equilibrium •Free Energy Change and the Equibrium Constant • = Standard State Free Energy Change = + RTln([products]/[reactants]) = + RTlnK, where K is equilbrium constant Free Energy Calculations •A Biochemical Example : •(1)G6P <=> F6P ( = +1.7 kJ/mol means equilibrium concentration has more G6P than F6P) ( 2 ) Plugging this into , one finds that (F6P)/(G6P) = 0.504 (3) Since G = + RTln([products]/[reactants]), displacements away from equilibrium will be moved towards equilibrium by corresponding force of free energy change brought about by the change. 2.7 How Cells Use Energy •Light from the sun is the ultimate source of energy for all life on earth –photosynthetic organisms use light energy to drive the energy-requiring synthesis of carbohydrates –non-photosynthetic organisms consume these carbohydrates and use them as energy sources 2.8 Common Ground for Cells •Eukaryotes are complex; how did such cells arise from simplest progenitors? •Mutualism: a symbiotic association between two organisms that gives rise to a new organism combining characteristics of both original types
  5. 5. –the lichen, which consists of a fungus and an alga –the root nodule system formed by a leguminous plant and anaerobic nitrogen-fixing bacteria –humans and bacteria such as Escherichia coli that live in the human intestinal tract Common Ground for Cells –a similar model can be proposed for the origin of chloroplasts –the fact that mitochondria and chloroplasts have their own DNA and their own apparatus for the synthesis of RNA and proteins supports this model •These proposed connections between prokaryotes and eukaryotes are not established with complete certainty •Still they provide an interesting framework from which to consider the reactions that take place in cells The root nodule(根瘤) •root nodule system formed by a leguminous plant and anaerobic nitrogen- fixing bacteria lichen(地衣) Escherichia coli(大肠杆菌)
  6. 6. Chapter 1 Carbohydrates •Carbohydrate: a polyhydroxyaldehyde or polyhydroxyketone, or a substance that gives these compounds on hydrolysis •Monosaccharide: a carbohydrate that cannot be hydrolyzed to a simpler carbohydrate •they have the general formula CnH2nOn, where n varies from 3 to 8 •aldose: a monosaccharide containing an aldehyde group •ketose: a monosaccharide containing a ketone group Monosaccharides •Monosaccharides are classified by their number of carbon atoms •There are only two trioses •aldo- and keto- are often omitted and these compounds are referred to simply as trioses; although this designation does not tell the nature of the carbonyl group, it at least tells the number of carbons Monosaccharides •Glyceraldehyde contains a stereocenter and exists as a pair of enantiomers Fischer Projections •Fischer projection: a two dimensional representation for showing the configuration of tetrahedral stereocenters •horizontal lines represent bonds projecting forward •vertical lines represent bonds projecting to the rear •the carbon atom at the intersection of the horizontal and vertical lines is not shown D,L Monosaccharides •According to the conventions proposed by Fischer •D-monosaccharide: a monosaccharide that, when written as a Fischer projection, has the -OH on its penultimate carbon on the right •L-monosaccharide: a monosaccharide that, when written as a Fischer projection, has the -OH on its penultimate carbon on the left The four aldotetroses •Enantiomers: stereoisomers that are mirror images •example: D-erythrose and L-erythrose are enantiomers •Diastereomers: stereoisomers that are not mirror images •example: D-erythrose and D-threose are diastereomers D,L Monosaccharides •Following are the two most common D-aldotetroses and the two most common D-aldopentoses and the three most common D-
  7. 7. aldohexoses. •Note that the third of these is an amino sugar •also shown is the most common 2-keto-D-hexose Cyclic Structure •Monosaccharides have -OH and C=O groups in the same molecule and exist almost entirely as five- and six-membered cyclic hemiacetals •anomeric carbon: the new stereocenter resulting from cyclic hemiacetal formation •anomers: carbohydrates that differ in configuration only at their anomeric carbons •Haworth projections •five- and six-membered hemiacetals are represented as planar pentagons or hexagons, as the case may be, viewed through the edge •most commonly written with the anomeric carbon on the right and the hemiacetal oxygen to the back right •the designation t - means that -OH on the anomeric carbon is cis to the terminal -CH2OH; O- means that it is trans •a six-membered hemiacetal ring is shown by the infix -pyran- pyran •a five-membered hemiacetal ring is shown by the infix -furan- -furan Conformational Formulas •five-membered rings are so close to being planar that Haworth projections are adequate to represent furanoses •for pyranoses, the six-membered ring is more accurately represented as a strain-free chair conformation •if you compare the orientations of groups on carbons 1, 2, 3, 4, and 5 in the Haworth and chair projections of t -D-glucopyranose, you will see that in each case they are up-down-up-down-up respectively Ascorbic Acid (Vitamin C) •L-Ascorbic acid (vitamin C) is synthesized both biochemically and industrially from D-glucose •L-Ascorbic acid is very easily oxidized to L-dehydroascorbic acid •both are physiologically active and are found in most body fluids Oxidation •Reducing sugar: one that reduces an oxidizing agent •oxidation of a cyclic hemiacetal form gives a lactone •when the oxidizing agent is Tollens’ solution, Ag precipitates as a silver mirror Reduction •The carbonyl group of a monosaccharide can be reduced to an hydroxyl
  8. 8. group by a variety of reducing agents, including H2/M and NaBH4 •reduction of the C=O group of a monosaccharide gives a polyhydroxy compound called an alditol Phosphoric Esters •Phosphoric esters are particularly important in the metabolism of sugars •phosphoric esters are frequently formed by transfer of a phosphate group from ATP Formation of Glycosides •Glycoside: a carbohydrate in which the -OH of the anomeric carbon is Glycoside replaced by -OR •those derived from furanoses are furanosides; those derived from furanosides pyranoses are pyranosides •glycosidic bond: the bond from the anomeric carbon to the -OR group Amino Sugars Disaccharides •Sucrose •table sugar; obtained from the juice of sugar cane and sugar beet •one unit of D-glucose and one unit of D-fructose joined by an o -1,2- glycosidic bond •Lactose •about 5% - 8% in human milk, 4% - 5% in cow’s milk •one unit of D-galactose and one unit of D-glucose joined by a o -1,4- glycosidic bond •Maltose •two units of D-glucose joined by an t -1,4-glycosidic bond Polysaccharides •Cellulose: the major structural component of plants, especially wood and plant fibers •a linear polymer of approximately 2800 D-glucose units per molecule joined by β-1,4-glycosidic bonds •fully extended conformation with alternating 180° flips of glucose units •extensive intra- and intermolecular hydrogen bonding between chains •Starch: the major storage polysaccharides of plants •Starch is used for energy storage in plants •a polymers of α-D-glucose units •amylose: continuous, unbranched chains of up to 4000 : α-D-glucose amylose units joined by u α-1,4-glycosidic bonds •amylopectin: a highly branched polymer consisting of 24-30 units of D- amylopectin glucose joined by g α-1,4-glycosidic bonds and branches created by
  9. 9. α-1,6-glycosidic bonds •amylases catalyze hydrolysis of α-1,4-glycosidic bonds •debranching enzymes catalyze the hydrolysis of d α-1,6-glycosidic bonds Figure 13.22 Branching in amylopectin and glycogen •Chitin: the major structural component of the exoskeletons of invertebrates, such as insects and crustaceans; also occurs in cell walls of algae, fungi, and yeasts •composed of units of N-acetyl-β-D-glucosamine joined by - β-1,4- glycosidic bonds •Bacterial cell walls: prokaryotic cell walls are constructed on the framework of the repeating unit NAM-NAG joined by β-1,4-glycosidic bonds Bacterial Cell Walls •The NAM-NAG polysaccharide is in turn cross-linked by small peptides •in Staphylococcus aureus, the cross link is a tetrapeptide •this tetrapeptide is unusual in that it contains two amino acids of the D- series, namely D-Ala and D-Gln •each tetrapeptide is cross linked to an adjacent tetrapeptide by a pentapeptide of five glycine units Figure The peptidoglycan of a bacterial cell wall Plant Cell Walls •consist largely of cellulose •also contain pectin which functions as an intercellular cementing material •pectin is a polymer of D-galacturonic acid joined by p -1,4-glycosidic bonds •the major nonpolysaccharide of cell walls, especially in woody plants, is lignin (next screen) Figure Plant Cell Walls - Lignin •Glycosaminoglycans: polysaccharides based on a repeating disaccharide where one of the monomers is an amino sugar and the other has a negative charge due to a sulfate or carboxylate group •heparin: natural anticoagulant •hyaluronic acid: a component of the vitreous humor of the eye and the lubricating fluid of joints •chondroitin sulfate and keratan sulfate: components of connective tissue Hyaluronic Acid Heparin
  10. 10. Glycoproteins •Glycoproteins contain carbohydrate units covalently bonded to a polypeptide chain •antibodies are glycoproteins •carbohydrates play a role as antigenic determinants, the portions of the antigenic molecule that antibodies recognize and to which they bond Blood Group Substances •Membranes of animal plasma cells have large numbers of relatively small carbohydrates bound to them •these membrane-bound carbohydrates act as antigenic determinants •among the first antigenic determinants discovered were the blood group substances •in the ABO system, individuals are classified according to four blood types: A, B, AB, and O •at the cellular level, the biochemical basis for this classification is a group of relatively small membrane-bound carbohydrates ABO Blood Classification •in type A, the nonreducing end is NAGal •in type B it is Gal •in type AB, both types are present •in Type O, neither of these terminal residues is present L-Fucose •L-fucose is synthesized biochemically from D-mannose
  11. 11. Chapter 2 Lipids and Membranes Lipids •Lipids: a heterogeneous class of naturally occurring organic compounds classified together on the basis of common solubility properties •insoluble in water, but soluble in aprotic organic solvents including diethyl ether, chloroform, methylene chloride, and acetone •Lipids include •fatty acids, triacylglycerols, sphingolipids, phosphoacylglycerols, glycolipids, •lipid-soluble vitamins •prostaglandins, leukotrienes, and thromboxanes •cholesterol, steroid hormones, and bile acids Fatty Acids •Fatty acid: an unbranched-chain carboxylic acid, most commonly of 12 - 20 carbons, derived from hydrolysis of animal fats, vegetable oils, or phosphodiacylglycerols of biological membranes •In the shorthand notation for fatty acids •the number of carbons and the number of double bonds in the chain are shown by two numbers, separated by a colon •Among the fatty acids most abundant in plants and animals •nearly all have an even number of carbon atoms, most between 12 and 20, in an unbranched chain •the three most abundant are palmitic (16:0), stearic (18:0), and oleic (18:1) acids •in most unsaturated fatty acids, the cis isomer predominates; the trans isomer is rare •unsaturated fatty acids have lower melting points than their saturated counterparts; the greater the degree of unsaturation, the lower the melting point Triacylglycerols •Triacylglycerol (triglyceride): an ester of glycerol with three fatty acids •natural soaps are prepared by boiling triglycerides (animal fats or vegetable oils) with NaOH, in a reaction called saponification (Latin, sapo, soap) Soaps •Soaps form water-insoluble salts when used in water containing Ca(II),
  12. 12. Mg(II), and Fe(III) ions (hard water) water Phosphoacylglycerols •Phosphoacylglycerols (phosphoglycerides) are the second most abundant group of naturally occurring lipids •found almost exclusively in plant and animal membranes, which typically consist of 40% -50% phosphoacylglycerols and 50% - 60% proteins •the most abundant phosphoacylglycerols are derived from phosphatidic acid, a molecule in which glycerol is esterified with two molecules of fatty acid and one of phosphoric acid •the three most abundant fatty acids in phosphatidic acids are palmitic (16:0), stearic (18:0), and oleic (18:1) •A phosphatidic acid •further esterification with a low-molecular-weight alcohol gives a phosphoacylglycerol •the most common of these low-molecular-weight alcohols are •A lecithin Waxes •An ester of a long-chain fatty acid and alcohol •from the Old English word weax = honeycomb Sphingolipids •contain sphingosine, a long-chain aminoalcohol from which this class is named Glycolipids •Glycolipid: a compound in which a carbohydrate is bound to an -OH of the lipid •many glycolipids are derived from ceramides Steroids •Steroids: a group of plant and animal lipids that have this tetracyclic ring structure •The features common to the ring system of most naturally occurring steroids are illustrated here Androgens •Androgens: male sex hormones •synthesized in the testes
  13. 13. •responsible for the development of male secondary sex characteristics Estrogens •Estrogens: female sex hormones •synthesized in the ovaries •responsible for the development of female secondary sex characteristics and control of the menstrual cycle Cholesterol •The steroid of most interest in our discussion of biological membranes is cholesterol Biological Membranes •In aqueous solution, phosphoglycerides spontaneously form into a lipid bilayer, with a back-to-back arrangement of lipid monolayers (see Figure) •polar head groups are in contact with the aqueous environment •nonpolar tails are buried within the bilayer •the major force driving the formation of lipid bilayers is hydrophobic interaction •the arrangement of hydrocarbon tails in the interior can be rigid (if rich in saturated fatty acids) or fluid (if rich in unsaturated fatty acids) Figure A lipid bilayer Biological Membranes •the presence of cholesterol increases rigidity •with heat, membranes become more disordered; the transition temperature is higher for more rigid membranes; it is lower for less rigid membranes •plant membranes have a higher percentage of unsaturated fatty acids than animal membranes •the presence of cholesterol is characteristic of animal rather than plant membranes •animal membranes are less fluid (more rigid) than plant membranes •the membranes of prokaryotes, which contain no appreciable amounts of steroids, are the most fluid Membrane Proteins •Functions: transport substances across membranes; act as receptor sites, and Functions sites of enzyme catalysis •Peripheral proteins •bound by electrostatic interactions •can be removed by raising the ionic strength
  14. 14. •Integral proteins •bound tightly to the interior of the membrane •can be removed by treatment with detergents or ultrasonification •removal generally denatures them Fluid Mosaic Model •Fluid: there is lateral motion of components in the membrane; Fluid •proteins, for example, “float” in the membrane and can move along its plane •Mosaic: components in the membrane exist side-by-side as separate entities Mosaic •the structure is that of a lipid bilayer with proteins, glycolipids, and steroids such as cholesterol embedded in it •no complexes, as for example, lipid-protein complexes, are formed Membrane Transport •Passive transport •driven by a concentration gradient •simple diffusion: a molecule or ion moves through an opening created by a channel protein •facilitated diffusion: a molecule or ion is carried across a membrane by a carrier protein •Active transport •a substance is moved against a concentration gradient •primary active transport: transport is linked to the hydrolysis of ATP or other high-energy molecule; for example, the Na+/K+ ion pump (Figures 7.24 and 7.25) •secondary active transport: driven by H+ gradient
  15. 15. Chapter 3 Amino Acids and Peptides Amino Acids •Amino acid: a compound that contains both an amino group and a carboxyl group •α-Amino acid: an amino acid in which the amino group is on the carbon adjacent to the carboxyl group •although α-amino acids are commonly written in the unionized form, they are more properly written in the zwitterion (internal salt) form Chirality of Amino Acids •With the exception of glycine, all protein-derived amino acids have at least one stereocenter (the α-carbon) and are chiral •the vast majority of α-amino acids have the L-configuration at the α- carbon •Comparison of the stereochemistry of alanine and glyceraldehyde (Fischer projection formulas) 20 Protein-Derived AA •Note these structural features 1. All 20 are α-amino acids 2. For 19 of the 20, the α-amino group is primary; for proline, it is secondary 3. With the exception of glycine, the α-carbon of each is a stereocenter 4. Isoleucine and threonine contain a second stereocenter 5. The sulfhydryl group (pKa 8.3) of cysteine, the imidazole group (pKa 6.0) of histidine, and the phenolic hydroxyl (pKa 10.1) of phenylalanine are partially ionized at pH 7.0, but the ionic form is not the major form at this pH Uncommon Amino Acids •Each example is derived from a common amino acid by the modification shown in color •hydroxylysine and hydroxyproline are found only in a few connective tissues such as collagen •thyroxine is found only in the thyroid gland Ionization of Amino Acids
  16. 16. Titration of Amino Acids Figure (a) Titration of alanine with NaOH Figure (b) Titration of histidine with NaOH Acidity: Aα-COOH Groups •The average pKa of an α-carboxyl group is 2.19, which makes them considerably stronger acids than acetic acid (pKa 4.76) •the greater acidity of the amino acid carboxyl group is due to the electron-withdrawing inductive effect of the -NH3+ group Acidity: α-NH3+ groups •The average value of pKa for an α-NH3 group is 9.47, compared with a + value of 10.76 for a 2° alkylammonium ion Basicity: Guanidine Group •The side chain of arginine is a considerably stronger base than an aliphatic amine •basicity of the guanido group is attributed to the large resonance stabilization of the protonated form relative to the neutral form Basicity: Imidazole Group •The imidazole group on the side chain of histidine is a heterocyclic aromatic amine • •Given the value of pKa of each functional group, we can calculate the ratio of each acid to its conjugate base as a function of pH •Consider the ionization of an α-COOH •writing the acid ionization constant and rearranging terms gives Ionization vs pH •substituting the value of Ka (1 x 10-2) for the hydrogen ion concentration at pH 7.0 (1.0 x 10-7) gives •at pH 7.0, the α-carboxyl group is virtually 100% in the ionized or conjugate base form, and has a net charge of -1 •we can repeat this calculation at any pH and determine the ratio of [w α- COO-] to [] α-COOH] and the net charge on the α-carboxyl at that pH •We can also calculate the ratio of acid to conjugate base for an α-NH3 + group; for this calculation, assume a value 10.0 for pKa •writing the acid ionization constant and rearranging gives •Ionization vs pH •substituting values for Ka of an α-NH3+ group and the hydrogen ion concentration at pH 7.0 gives •at pH 7.0, the ratio of α-NH2 to α-NH3 + is approximately 1 to 1000
  17. 17. •at this pH, an α-amino group is 99.9% in the acid or protonated form and has a charge of +1 Henderson-Hasselbalch •We have calculated the ratio of acid to conjugate base for an α-carboxyl group and an α-amino group at pH 7.0 •We can do this for any weak acid and its conjugate base at any pH using the Henderson-Hasselbalch equation •using the Henderson-Hasselbalch equation, we can calculate the percent of charged or uncharged form present and the net charge on serine at pH 3.0, 7.0, and 10.0 Isoelectric pH(pI) •Isoelectric pH, pI: the pH at which the majority of molecules of a compound in solution have no net charge •the pI for glycine, for example, falls midway between the pKa values for the carboxyl and amino groups •given in the following tables are isoelectric pH values for the 20 protein-derived amino acids Electrophoresis •Electrophoresis: the process of separating compounds on the basis of their electric charge •electrophoresis of amino acids can be carried out using paper, starch, agar, certain plastics, and cellulose acetate as solid supports •in paper electrophoresis, a paper strip saturated with an aqueous buffer of predetermined pH serves as a bridge between two electrode vessels Electrophoresis(Process) •a sample of amino acids is applied as a spot (the origin) on the solid support strip •an electric potential is applied to the electrode vessels and amino acids migrate toward the electrode with charge opposite their own •molecules with a high charge density move faster than those with a low charge density •molecules at their isoelectric point remain at the origin •after separation is complete, the strip is dried and developed to make the separated amino acids visible Polypeptides •In 1902, Emil Fischer proposed that proteins are long chains of amino acids joined by amide bonds to which he gave the name peptide bonds
  18. 18. •Peptide bond: the special name given to the amide bond between the - carboxyl group of one amino acid and the c -amino group of another Serylalanine (Ser-Ala) Peptides •peptide: the name given to a short polymer of amino acids joined by peptide peptide bonds; they are classified by the number of amino acids in the chain •dipeptide: a molecule containing two amino acids joined by a peptide dipeptide bond •tripeptide: a molecule containing three amino acids joined by peptide tripeptide bonds •polypeptide: a macromolecule containing many amino acids joined by polypeptide peptide bonds •protein: a biological macromolecule of molecular weight 5000 g/mol or protein greater, consisting of one or more polypeptide chains Geometry of Peptide Bond •the four atoms of a peptide bond and the two alpha carbons joined to it lie in a plane with bond angles of 120° about C and N •to account for this geometry, Linus Pauling proposed that a peptide bond is most accurately represented as a hybrid of two contributing structures •the hybrid has considerable C-N double bond character and rotation about the peptide bond is restricted Writing Peptides •By convention, peptides are written from the left, beginning with the free -NH3+ group and ending with the free -COO- group •the repeat pattern, starting from the N-terminal amino acid, is N ---> t - carbon ---> carbonyl carbon etc. Some Small Peptides Glutathione Enkephalins Oxytocin & Vasopressin
  19. 19. Chapter 4 Proteins Protein Structure •1° structure: the sequence of amino acids in a polypeptide chain, read from the N-terminal end to the C-terminal end •2° structure: the ordered 3-dimensional arrangements (conformations) in localized regions of a polypeptide chain; refers only to interactions of the peptide backbone •e. g., e α-helix and - β-pleated sheet Ramachandran Angles R α-Helix - coil of the helix is clockwise or right-handed •there are 3.6 amino acids per turn •repeat distance is 5.4Å •each peptide bond is s-trans and planar •C=O of each peptide bond is hydrogen bonded to the N-H of the fourth amino acid away •C=O----H-N hydrogen bonds are parallel to helical axis •all R groups point outward from helix a α-Helix •Several factors can disrupt an α-helix •proline creates a bend because of (1) the restricted rotation due to its cyclic structure and (2) its α-amino group has no N-H for hydrogen bonding •strong electrostatic repulsion caused by the proximity of several side chains of like charge, e.g., Lys and Arg or Glu and Asp •steric crowding caused by the proximity of bulky side chains, e.g., Val, Ile, Thr I β-Pleated Sheet - polypeptide chains lie adjacent to one another; may be parallel or antiparallel •R groups alternate, first above and then below plane •each peptide bond is s-trans and planar •C=O and N-H groups of each peptide bond are perpendicular to axis of the sheet
  20. 20. •C=O---H-N hydrogen bonds are between adjacent sheets and perpendicular to the direction of the sheet β -TURN T α-Helices and - β-Sheets •Supersecondary structures: the combination of α- and - β-sections, as for example •• β αβα unit: two parallel strands of β-sheet connected by a stretch of α α-helix •• α α unit: two antiparallel α-helices α •• β-meander: an antiparallel sheet formed by a series of tight reverse α turns connecting stretches of a polypeptide chain •Greek key: a repetitive supersecondary structure formed when an antiparallel sheet doubles back on itself •• β-barrel: created when β-sheets are extensive enough to fold back G on themselves Collagen Triple Helix •consists of three polypeptide chains wrapped around each other in a ropelike twist to form a triple helix called tropocollagen; MW approx. 300,000 •30% of amino acids in each chain are Pro and Hyp (hydroxyproline); hydroxylysine also occurs •every third position is Gly and repeating sequences are X-Pro-Gly and X-Hyp-Gly •each polypeptide chain is a helix but not an e α-helix •the three strands are held together by hydrogen bonding involving hydroxyproline and hydroxylysine •with age, collagen helices become cross linked by covalent bonds formed between Lys and His residues •deficiency of Hyp results in fragile collagen Factors Determining Secondary and Tertiary Structure Ramachandran Plots 3° and 4° Structure •Tertiary (3°) structure: the arrangement in space of all atoms in a polypeptide chain •it is not always possible to draw a clear distinction between 2°and 3°
  21. 21. structure •Quaternary (4°) structure: the association of polypeptide chains into aggregations •Proteins are divided into two large classes based on their three-dimensional structure •fibrous proteins •globular proteins Fibrous Proteins •Fibrous proteins: contain polypeptide chains organized approximately parallel along a single axis. They •consist of long fibers or large sheets •tend to be mechanically strong •are insoluble in water and dilute salt solutions •play important structural roles in nature •Examples are •keratin of hair and wool •collagen of connective tissue of animals including cartilage, bones, teeth, skin, and blood vessels keratins fibroin Collagen in connective tissue Elastin and desmosine Globular Proteins •Globular proteins: proteins which are folded to a more or less spherical proteins shape •they tend to be soluble in water and salt solutions •most of their polar side chains are on the outside and interact with the aqueous environment by hydrogen bonding and ion-dipole interactions •most of their nonpolar side chains are buried inside •nearly all have substantial sections of n α-helix and - β-sheet •Examples are •myoglobin (Figure) •hemoglobin Factors Directing Folding •Noncovalent interactions, including •hydrogen bonding between polar side chains, e.g., Ser and Thr
  22. 22. •hydrophobic interaction between nonpolar side chains, e.g., Val and Ile •electrostatic attraction between side chains of opposite charge, e.g., Lys and Glu •electrostatic repulsion between side chains of like charge, e.g., Lys and Arg, Glu and Asp •Formation of disulfide (-S-S-) bonds between side chains of cysteines Factors Determining Tertiary Structure •BPTI 3° Structure •x-ray crystallography •uses a perfect crystal; that is, one in which all individual protein molecules have the same 3D structure and orientation •exposure to a beam of x-rays gives a series diffraction patterns •information on molecular coordinates is extracted by a mathematical analysis called a Fourier series •2-D Nuclear magnetic resonance •can be done on protein samples in aqueous solution Quaternary Structure •Quaternary (4°) structure: the association of polypepetide monomers into multisubunit proteins •examples we will see in this course Denaturation •Denaturation: the loss of the structural order (2°, 3°, 4°, or a combination of these) that gives a protein its biological activity; that is, the loss of biological activity •Denaturation can be brought about by •heat •large changes in pH, which alter charges on side chains, e.g., -COO- to - COOH or -NHC + to -NH •detergents such as sodium dodecyl sulfate (SDS) which disrupt hydrophobic interactions •urea or guanidine, which disrupt hydrogen bonding •mercaptoethanol, which reduces disulfide bonds Protein Folding Chaperones •primary structure conveys all information necessary to produce the correct 3° structure
  23. 23. •nonetheless, in the protein-dense environment of a cell, proteins may begin to fold incorrectly or may associate with other proteins before folding is completed •special proteins called chaperones aid in the correct and timely folding of many proteins •hsp70 were the first protein chaperone discovered •chaperones exist in organisms from prokaryotes to humans chaperone •Hemoglubin and Immunoglobulins Myoglobin Structure of Myoglobin •a single polypeptide chain of 153 amino acids •a single heme group in a hydrophobic pocket •8 regions of α-helix; no regions of - β-sheet •most polar side chains are on the surface •nonpolar side chains are folded to the interior •two His side chains are in the interior, involved with interaction with the heme group •Fe(II) of heme has 6 coordinates sites; 4 interact with N atoms of heme, 1 with N of a His side chain, and 1 with either an O 2 molecule or an N of the second His side chain Heme structure Hemoglobin Oxygen Binding of Hb •a tetramer of two α-chains (141 amino acids each) and two - β-chains (153 amino acids each); ( •each chain has 1 heme group; hemoglobin can bind up to 4 molecules of O2 •binding is cooperative; when one O2 is bound, it becomes easier for the next O2 to bind •the function of hemoglobin is to transport oxygen •the structure of oxygenated Hb is different from that of unoxygenated Hb •H+, CO2, Cl-, and 2,3-bisphosphoglycerate (BPG) affect the ability of Hb to bind and transport oxygen Oxygen Binding of Hb Fig : O2 binding of hemoglobin and myoglobin
  24. 24. Cooperativity of Binding/Release The oxygenation state (filled or empty) of one site of the multisubunit hemoglobin can be communicated to another site, resulting in cooperative binding and release of oxygen. —— Allosteric binding Oxygen Binding of Hb •The effect of pH on the oxygen-binding ability of Hb is called the Bohr effect as pH decreases (more acidic), oxygen is released •CO2 promotes release of O2 from HbO2 Oxygen Binding of Hb Figure The Bohr effect Table Summary of the Bohr effect Hemoglobin (Hb) •Hemoglobin in blood is bound to BPG •interaction is electrostatic, between negative charges on BPG(2,3- bisphosphoglycerate) and positive side chains (e.g., Lys, Arg) of hemoglobin •BPG promotes O2 dissociation •Hb stripped of BPG remains saturated with O2 Fetal Hemoglobin, Hb F •has a higher affinity for O2 than maternal Hb A •structure is s α2β2 •binds less strongly to BPG that does Hb A Figure: Oxygen binding capacity of Hb F Abnormal Human Hb( Hb Variants) •Hb S: substitution of Val for Glu at 26H •Hb E: Glu B8(26)H -> Lys; change is on the surface and has little effect on Hb stability or function •Hb Savannah: Gly B6(24)H -> Val; not enough room for Val between B-helix and E-helix which disrupts entire structure •Hb Bibba: Leu H19(136)H -> Pro; proline disrupts the H-helix •Hb M Iwate: His F8(87)H -> Tyr; blood contains methemoglobin and blood is chocolate brown •Hb Milwaukee: Val E11(67)H -> Glu; glutamate side chain forms an ion pair with heme iron which stabilizes Fe(III) and prevents O2 binding
  25. 25. •Fig Evolution of Myoglobin / Hemoglobin Proteins •Out of the 153 amino acids in the amino acid sequences of sperm whale myoglobin and human myoglobin, there are only 25 differences.(∼100 million years) •Conserved Amino Acid Sequences - During the long evolution of the myoglobin/hemoglobin family of proteins, only a few amino acid residues have remained invariant . Immuglobulins •Antigens and Antibodies - The foreign substance that elicits an immune response is called the antigen. A specific immunoglobulin that binds to the antigen is called the antibody. •1.Humoral immune response - Lymphatic cells called B lymphocytes synthesize specific immunoglobulin molecules that are excreted from the cell and bind to the invading substance. Binding either precipitates the foreign substance or marks it for destruction by cells called macrophages. •2. Cellular immune response - Lymphatic cells called T lymphocytes, bearing immunoglobulin-like molecules on their surfaces, recognize and kill foreign or aberrant cells. Terms •Antigen: Foreign material that is recognized by the immune system, it is usually a protein, but it can be a peptide, or carbohydrate. •Epitope: Region of a protein antigen to which the antibody binds. •Hapten: A small chemical that is an antigen. Antibody Structure: •1 Quaternary structure (2 Light + 2 Heavy chains). The two heavy and light chains are held together by non-covalent forces and covalent (disulfide) bonds. The light chain consists of two immunoglobulin folds and the heavy chain contains four of these domains: The overall shape is that of a ‘Y’. Two antigen binding sites/antibody. Antibody structure •2 Immunoglobulin fold is an example of a protein domain or a motif. It contains 7 β -strands that form a two sheet sandwich with 4 stands on one side and 3 on the other. A buried disulfide bond crosslinks the two faces. Antibody structure
  26. 26. •3 Disulfide bonds covalently join the heavy and light chains, conferring stability on this secreted protein (Some antibodies are secreted outside the body) •4 hypervariable regions(CDR (Complementary determining region)) •The first immunoglobulin domain of the heavy and light chain contains three special segments of primary sequence that vary in their primary sequence from one antibody to the next. Antibody structure •Fab fragments can be further reduced to Fv fragments, consisting of the 1st immunoglobulin fold from the heavy and light chain. The Fv domain is the smallest unit that can bind antigen. Practical Uses of Immunoglobulins: •Fluorescence tagging (to label various components in the cell) •Purification of materials (More on this later) •Immunotherapy (see Campbell) •Novel chemical reactions (Some antibodies can actually perform chemical reactions) •Drug detoxification, see Chime page on Antibodies and Angel dust (PCP) Problem and practice Protein G •Protein G is a multidomain extracellular protein found in several Streptococcal species. The “protein G” referred to in this quiz is actually only the B1 domain of one of these proteins. These repeated domains bind to the Fc portion of IgG. There is no structural similarity between these domains and the StrepA protein, which has similar binding properties. 蛋白 质G是在几种链球菌中发现的一种多域的胞外蛋白质.此处展示的只是 其“B1”域。B1域与IgG的Fc部分结合 Structure of Protein G (front view and side view) •1. protein G amino terminus? •2. protein G carboxyl terminus? Structure of Protein G(back view) •3. β -hairpin motif?(2) •4. a type I (or "common") turn?
  27. 27. •Which is type I turn that connects β-strands at the carboxyl-terminus Structure of Protein G(side view) •5. hydrophilic surface of the α-helix? •6. positive end of an α -helical dipole? •7. hydrophobic surface on β -strands? Problems about Structure of Protein G •1. A portion of the protein G -sheet is shown without the side-chains and with the backbone displayed as Sticks. Hydrogen bonds are indicated as dashed lines between the β-strands. Note that the H-bonds on the left are perpendicular to the chain direction, whereas those on the right are more evenly-spaced, but are set at an angle to the chain direction. •The H-bonds between the parallel –strands? •2. A segment of the α-helix backbone is shown as Sticks. The Glu27 residue is shown as Ball and Stick and the other side-chains are Wireframe. •Click on the atom that donates a hydrogen bond to the backbone C=O of Glu27 •3.Three residues near the N-terminus of protein G are shown as Ball and Stick: Phe52, Thr53, and Val54. •The peptide bond between Thr53 and Val54? •4.Three residues in the α-helix of protein G are shown as Ball and Stick: Ala24, Thr25, and Ala26. Helical geometry is fixed by preferred dihedral angles about these two backbone bonds: (Psi), rotation about the C -C single bond; and (Phi), rotation about the N-C single bond. •The φ or ϕ bond of the Thr25 residue? •5. The display is Spacefill with Structure coloring except that the -helix backbone is shown as Ball and Stick (with a magenta spiral tracing the helix). The location of Thr25 near the N-terminus of the helix is labelled. •Pick an α-helical residue about 10Å from Thr25. •6. A segment of the -helix backbone is shown as Sticks. The Glu27 residue is shown as Ball and Stick and the other side-chains are Wireframe. •Click on the atom that accepts a hydrogen bond from the N-H of Lys28.
  28. 28. •7.The display is Spacefill with Structure coloring except that three strands of the β -sheet are shown as Sticks. One strand is shown as Ball and Stick with Thr2 near the N-terminus labeled. •Pick a residue on the same β-strand as, and about 15Å from Thr2. Structure of Protein G •1. The C-terminal β-hairpin structure is shown with the sidechains of five threonines displayed as Ball and Stick. Distances (in Å units) between three pairs of these residues are shown as dashed red lines. •Pick the most likely of these potential H-bonds that also occurs between residues on the same β-strand. •2.The sidechains on the solvent- exposed surface of the β-sheet are shown as Ball and Stick. The remainder of protein G is Wireframe with Structure coloring. Most of the sidechains here are polar. In addition, two are basic. •Pick one of these basic sidechains. •3. The backbone is shown as Sticks with Structure coloring. The sidechains of the aromatic residues (Tyr, Trp, and Phe) are displayed as Ball and Stick and colored CPK. •Click on the residue that is responsible for most of the UV absorbance at 280 nm. •4. The sidechains on the hydrophilic surface of the β-sheet are shown as Spacefill. As noted in a previous question, nearly all of these sidechains are polar. In fact, Ile6 is the only apolar sidechain on the solvent-exposed surface of the β-sheet. •Click on Ile6. •5. The sidechains of the six aromatic residues are shown as Spacefill. This side view shows how they fill the hydrophobic core between the α -helix and β-sheet of protein G. •Pick a Phe residue. •6.The sequence of protein G is shown using the one-letter amino acid code. The segments corresponding to secondary structures are indicated with RasMol Structure coloring. •From the groups of five amino acids listed below the sequence, pick the group that is not found in protein G.
  29. 29. • •7. Protein G is shown in backbone display with RasMol Structure coloring. But someone has tampered with this picture! It shows an impossible path of the polypeptide chain. •Pick the position where this impossible backbone topology occurs.
  30. 30. Chapter 5 Neucleic Acids • •5.1 Two Nucleic Acids: DNA and RNA •5.2 Propertie of nucleotides 核苷酸 •5.3 Primary Structure of Nucleic Acids 核酸的一级结构 •5.4 Secondary Structure and tertiary Structure of Nucleic Acids 核酸的二 级结构和三级结构 •5.5 Stability of Secondary and Tertiary Structure核酸的二级结构和三级结 构的稳定性 5.2 Nucleotides Nucleotides = Base + Sugar + Phosphate Nucleoside= Sugar + Base (no phosphate) Standard Nucleotides: •5’- adenosine monophosphate 5’-AMP ( 5’-腺苷酸) •5’- guanosine monophosphate 5’-GMP ( 5’-鸟苷酸) •5’- cytidine monophosphate 5’-CMP ( 5’-胞苷酸) •5’- uridine monophosphate 5’-UMP ( 5’-脲苷酸) •5’- deoxyadenosine monophosphate 5’-dAMP • ( 5’ -脱氧腺苷酸) •5’- deoxyguanosine monophosphate 5’-dGMP •(5’-脱氧鸟苷酸) •5’- deoxycytidine monophosphate 5’-dCMP (5’-脱氧胞苷酸) •5’-deoxythymidine monophosphate) 5’-dTMP •(5’-脱氧胸腺苷酸 Minor Bases or Modified bases (稀有碱基或修饰碱基) Nucleotide polyphosphate多磷酸核苷 •8 NDPs ( 5’-nucleotide diphosphate 5’-核苷二磷酸) •8 NTPs ( 5’-nucleotide triphosphate 5’-核苷三磷酸) Cyclic Nucleotides CoA、NAD + (P)and FAD •Co-enzyme A(CoA) •Nicotinamide adenine dinucleotide (NAD+)烟酰胺腺嘌呤二核苷酸、 •Flavin Adenine Dinucleotide (FAD)黄素腺嘌呤二核苷酸
  31. 31. NAD NADP FAD Nucleotide propertics 1. Dissociation in water •The phosphate group of a nucleotide acts as a strong acid (pKa 1), •The amine groups of the purine and pyrimidine bases can be protonated. 2.Tautomerization of the bases •The bases can tautomerize(互变异构); that is, the bases can redistribute positions of hydrogens and double bonds Unusual base pair Nucleotide propertics 3. Spectrum •Nucleotides absorb light strongly in the near-ultraviolet region of the spectrum. ε260(X104) AMP 1.54 GMP 1.17 CMP 0.75 dTMP 0.92 UMP 0.99. Biological functions of nucleotides •1、The monomeric units of nucleic acids •2、Energy carrier:ATP、GTP、CTP、UTP •3、signals :cAMP、cGMP •4、coenzymes:FAD、NAD、CoA 5.3 Primary Structure of Nucleic Acids •The primary structure is the sequence of nucleoside monophosphates •Directionality :5’→3’ •Individuality Significance of Primary Structure •Genetic information stored in nucleotide sequence of DNA
  32. 32. •Gene is a particular DNA sequence DNA is genetic substance. Hershey and Chase - Bacteriophage T2 transfers DNA to bacteria 5.4 Secondary structure of DNA •The secondary structure refers to the shape a nucleic acid assumes as a result of the primary structure. •B-DNA : predominant form in the aqueous environment of the cell •A-DNA •Z-DNA : purine/pyrimidine tracts Secondary structure of DNA Structure of B-DNA — The two chains in the double helix are antiparallel — Phosphate groups link together the sugar backbone via phosphodiester bonds. — The bases on the two chains pair in a complementary fashion. A=T,G=C — Hydrogen bonds between bases hold the double helix together. (A=T,G≡C) —B-DNA:d= 2 nm,pitch = 3.4 nm , 10 bp / turn of the helix DNA base pair DNA base pair DNA base pair The discovery of DNA double helix •Chargaff's Rule (A=T, G=C in DNA) •Franklin, Wilkins: X-ray Diffraction Refined Structure Watson, Crick Significance of DNA double helix Meselson and Stahl experiment •1958 Application of Complementarity •Molecule hybridization Unusual Secondary Structures of DNA •Palindromes(回文结构)回Cruciform formation in palindrome sequences
  33. 33. GGCGCGCC CCGCGCGG H-DNA •purines/pyrimidines on one strand 5.5 Tertiary structure of DNA and RNA •Tertiary structure refers to large-scale folding in a linear polymer that is at a higher order than secondary structure. The tertiary structure is the specific three-dimensional shape into which an entire chain is folded. Tertiary structure of DNA Circular DNA and Supercoiling • Circular DNAs •Supercoiling : •Positive(+)----additional twists added beyond the normal amount for linear DNA •negative (-)----reduced numbers of twists compared to linear DNA. Supercoiling •Twists(T, 缠绕数): The total number of times the two strands of the helix cross over each other, excluding writhing. Twist is a measure of how tightly the helix is wound. •Writhes (W,扭曲数): The number of times the helix as a whole crosses over itself - that is, the number of superhelical turns that are present •Linking number (L , 连接 数): The total number of times two strands of a DNA helix cross each other by means of either twist or writhe L=T+W •The superhelical density : L/L0, where L0 is the linking number of the DNA in its unstrained (relaxed state). •Besides writhing, unwinding DNA, cruciform formation (via palindromes), triple helix formation (H-DNA), and Z-DNA formation, can all reduce superhelical tension, too. Ethidium bromide(溴乙锭)change DNA supercoil problem set RNA
  34. 34. tRNA 4.6 The Biological Functions of Nucleic Acids •1、Genome •2、Transcription •3、Translation: •rRNA(ribosome: Ribozyme, Structure) •mRNA •tRNA 5.7 Stability of Nucleic Acid Secondary and Tertiary Structure •Stability and Denaturation Denaturation: (1) The electrostatic repulsion of the negative charges on the phosphate groups. (2)The higher entropy of the denatured state (the denatured form has more possible conformations than the double helix form, so the denatured form has greater randomness). Stability: (1)hydrogen bonds (2) The van der Waals interactions between the planar bases Factors favoring dissociation of double helices to random coils 1 、 Hypochromism - absorption of light by bases reduced when in helix = denaturation causes increase in absorbance of light at 260 nm. Melting Temperature(Tm): •The factors affecting on Tm: •①G-C(0.15mol/LNaCl//0.15mol/L sodium citrate) Tm=69.3+0.41(G+C) ②DNA purity ③pH, µ
  35. 35. Chapter 6 Enzymes : A Biological Catalyst Important Features of Enzyme Catalysis 1. With the exception of some RNAs that catalyze their own splicing (Section 10.4), all enzymes are proteins 2. Enzymes can increase the rate of a reaction by a factor of up to 1020 over an uncatalyzed reaction 3. Some enzymes are so specific that they catalyze the reaction of only one stereoisomer; others catalyze a family of similar reactions 4. Enzymes do not change the equilibrium point of reactions. 5. Enzymes are not changed by the overall reaction. (Although they may be modified during the reaction) Important Features of Enzyme Catalysis 6 .Catalysis occurs at the "active site", which is specific for certain substrates. The active site has the following with respect to its substrate: •Geometric complementarity; •Energetic complementarity. 7. Enzyme activity is regulated: •at genetic level by changes in protein production (transcription, translation); •by concentration of substrate(s) and product(s); •by the presence of allosteric activators and inhibitors. •by proteolytic cleavage of zymogens. Enzyme Kinetics •Initial rate of an enzyme-catalyzed reaction Activation Energy •The rate of a reaction depends on its activation energy, T G°‡ •an enzyme provides an alternative pathway with a lower activation energy Activation Energy Profile An enzyme alters the rate of a reaction, but not its free energy change or position of equilibrium Activation Energy calculation •For a reaction taking place at constant temperature and pressure, e.g., in the body
  36. 36. the change in free energy is •The change in free energy is related to the equilibrium constant, Keq, for the reaction by Activation Energy of Enzyme Catalysis •Consider the reaction Active site of Enzyme •In an enzyme-catalyzed reaction •substrate, S: a reactant •active site: the small portion of the enzyme surface where the substrate(s) becomes bound by noncovalent forces, e.g., hydrogen bonding, electrostatic attractions, van der Waals attractions Active site of Enzyme Enzyme-substrate complex •Two models have been developed to describe formation of the enzyme- substrate complex •lock-and-key model: substrate binds to that portion of the enzyme with a complementary shape •induced fit model: binding of the substrate induces a change in the conformation of the enzyme that results in a complementary fit Enzyme -Transition state Figure Activation energy profile for formation of an E-S complex Example of Enzyme Catalysis •Chymotrypsin catalyzes the selective hydrolysis of peptide bonds where the carboxyl is contributed by Phe and Tyr •it also catalyzes hydrolysis of the ester bond of p-nitrophenyl esters Chymotrypsin Enzyme activity is regulated •Aspartate transcarbamylase (ATCase) catalyzes this reaction ATCase Enzymes Kinetics Velocity of Reaction •For the reaction –the rate of reaction is given by rate equation –where k is a proportionality constant called the specific rate constant
  37. 37. –order of reaction: the sum of the exponents in the rate equation Velocity of Enzyme Catalysis –consider the reaction whose rate equation is given by the expression the reaction is said to be first order in A, first order in B, and second order overall –consider this reaction of glycogen with phosphate Enzyme saturation •Initial rate of an enzyme-catalyzed reaction versus substrate concentration Michaelis-Menten Model –for an enzyme-catalyzed reaction –the rates of formation and breakdown of ES are given by these equations –at the steady state –when the steady state is reached, the concentration of free enzyme is the total less that bound in ES –substituting for the concentration of free enzyme and collecting all rate constants in one term gives –where KM is called the Michaelis constant –it is now possible to solve for the concentration of the enzyme-substrate complex, [ES] –or alternatively –in the initial stages, formation of product depends only on the rate of breakdown of ES –if substrate concentration is so large that the enzyme is saturated with substrate [ES] = [E]T –substituting k2[E]T = Vmax into the top equation gives –when [S]= KM, the equation reduces to –it is difficult to determine Vmax experimentally –the equation for a hyperbola –can be transformed into the equation for a straight line by taking the reciprocal of each side Lineweaver-Burk Plot –which has the form y = mx + b, and is the formula for a straight line
  38. 38. –a plot of 1/V versus 1/[S] will give a straight line with slope of KM/Vmax and y intercept of 1/Vmax –such a plot is known as a Lineweaver-Burk double reciprocal plot Lineweaver-Burk Plot –KM is the dissociation constant for ES; the greater the value of KM, the less tightly S is bound to E –Vmax is the turnover number Turnover Numbers and KM Table 5.2 Values for some typical enzymes Enzyme Kinetics – Inhibitors Signification •1. Mechanistic studies to learn about how enzymes interact with their substrates. •2. Role of inhibitors in enzyme regulation. •3. Drugs if they inhibit aberrant biochemical reactions: • methotrexate: anti-cancer drug that affects DNA metabolism in actively growing cells • viagra: interfers with nitrous oxide chemistry (NO is a vasodialator) •penicillin, ampicillin, etc.:interfere with the synthesis of bacterial cell walls •4. Understanding the role of biological toxins. • Arsenate - mimics phosphate esters in enzyme reactions, but are easily hydrolysed. • Amino acid analogs - useful herbicides (i.e. roundup) • Insecticides - chemicals targeted for insect nervous system. Enzyme Inhibition •Reversible inhibitor: a substance that binds to an enzyme to inhibit it, but can be released –competitive inhibitor: binds to the active (catalytic) site and blocks access to it by substrate –noncompetitive inhibitor: binds to a site other than the active site; inhibits the enzyme by changing its conformation •Irreversible inhibitor: a substance that causes inhibition that cannot be reversed –usually involves formation or breaking of covalent bonds to or on the enzyme
  39. 39. •Allosteric Control - non-competitive inhibitors are allosteric inhibitors and activators. Competitive Inhibition –substrate must compete with inhibitor for the active site; more substrate is required to reach a given reaction velocity –we can write a dissociation constant, KI for EI –in a Lineweaver-Burk double reciprocal plot of 1/V versus 1/[S], the slope (and the x intercept) changes but the y intercept does not change  Inhibitor binds to the same site on the enzyme as the substrate.  Inhibitor ONLY binds the the free enzyme.  Inhibitor usually is structurally very similar to the substrate. For example, succinate is the normal substrate for the enzyme succinate dehydrogenase. Malonate is an effective competitive inhibitor of this enzyme.  VMAX is unchanged: At high levels of substrate all of the inhibitor can be displaced by substrate  KM is increased: It requires more substrate to reach 1/2 maximal velocity Noncompetitive Inhibition –several equilibria are involved –the maximum velocity VImax has the form –because the inhibitor does not interfere with binding of substrate to the active site, KM is unchanged –increasing substrate concentration cannot overcome noncompetitive inhibition Allosteric inhibition •Allosteric Activators increase substrate binding and/or the rate of the chemical step (kcat). •Allosteric inhibitors reduce substrate binding and/or the rate of the chemical step. Allosteric Enzymes •Allosteric: Greek allo + steric, other shape •Allosteric enzyme: an oligomer whose biological activity is affected by other substances binding to it
  40. 40. –these substances change the enzyme’s activity by altering the conformation(s) of its 4° structure •Allosteric effector: a substance that modifies the behavior of an allosteric enzyme; may be an –allosteric inhibitor –allosteric activator •Aspartate transcarbamoylase (ATCase) –feedback inhibition Enzyme Mechanism: Serine Proteases Active sites of enzymes •It is an area of enzyme, •It is consist of a few amino acids residues, •The residues are closed to each other in space •It combines substrate and catalyzes a reaction. Key Terms: •Covalent catalysis •Acyl-enzyme intermediate •Nucleophilic agent •Electrophilic agent •Preferential binding of transition state complex •Catalytic triad Diverse Role of Serine proteases •Serine proteases play an important role in many processes, e.g. digestion of dietary protein, blood clotting cascade, and in several pathways of differentiation and development. •Proteases active in digestion include: Trypsin , Chymotrypsin , Elastase •These enzymes are produced as zymogens. Proteolytic cleavage occurs in several locations resulting in the formation of intact enzymes. The active site in the zymogen is distorted and does not have high catalytic efficiency chymotrypsin Substrate Specificity •Serine proteases utilize all of the intermolecular forces. In particular, the
  41. 41. peptide to be cleaved often forms one or more hydrogen bonds with the substrate: •Trypsin cleaves after Lys and Arg residues: Asp189 in the active site of Trypsin interacts with the positive charge on Arg and Lys. •Chymotrypsin cleaves after aromatic (and large hydrophobic) residues: The active site of the enzyme contains a hydrophobic pocket, formed in part by Met192 of Chymotrypsin. Mechanism of Serine Proteases : •Serine Proteases can hydrolyze either esters or peptide bonds utilizing mechanisms of covalent catalysis and preferential binding of the transition state and by providing suitable reactive groups near the peptide bond that is cleaved. Ester Hydrolysis • •Note that the bright yellow color of the p-nitrophenolate ion provides a convenient way to monitor the rate of product formation. Peptide Hydrolysis •Chymotrypsin: Chemical Mechanism of Chymotrypsin •A key feature of the mechanism is the formation of a enzyme-bound stable Intermediate, or acyl-enzyme. Key residues are Ser195, His57, and Asp102: The catalytic triad. •I. Substrate binds •II. Nucleophilic attack of the side chain oxygen of Ser 195 on the carbonyl carbon of the scissile bond (bond to be cleaved) forming a tetrahedral intermediate. Assist from His 57 (proton transfer from Ser 195). Tetrahedral- intermediate is stabilized by amides of Ser195 and Gly 193. Chemical Mechanism of Chymotrypsin •III: Breakage of the peptide bond with assistance from His 57 (proton transfer to the new amino terminus). Release of the first product. •IV: Acyl-intermediate: Note that the substrate is attached to the active site Serine 195 •V: Nucleophilic attack of water on the acyl-enzyme intermediate with assistance of His 57 and formation of the tetrahedral intermediate.
  42. 42. •VI: Decomposition of acyl intermediate and release of the second product. Enzyme is in the same form as in panel I! Enzyme Regulation Allosteric regulation Covalent Modifications Aspartate transcarbamoylase ATCase •Organization of ATCase –catalytic unit: 6 subunits organized into 2 trimers –regulatory unit: 6 subunits organized into 3 dimers ATCase in metabolic pathway ATCase’s kinetics (a) Rate of ATCase catalysis vs substrate conc. (b) ATCase catalysis in presence of CTP; ATP ATCase’s allosteric effectors Why ? •The key to allosteric behavior is the existence of multiple forms for the 4º structure of the enzyme. •Allosteric effector modifies the 4º structure of an allosteric enzyme. Allosteric enzymes •Allosteric enzyme: an oligomer whose biological activity is affected by other substances binding to it –these substances change the enzyme’s activity by altering the conformation(s) of its 4° structure •Homoallostery: Binding of one substrate favors binding of additional substrates. •Heteroallostery :The kinetics of the enzyme can be controlled by any other substance that, in binding to the protein. •Allosteric effector: a substance that modifies the behavior of an allosteric enzyme; may be an –Allosteric Activators(positive effectors)increase substrate binding and/or the rate of the chemical step (kcat). –Allosteric inhibitors(negative effectors) reduce substrate binding and/or the rate of the chemical step. Allosteric Effects
  43. 43. –homotropic effects: allosteric interactions that occur when several identical molecules are bound to the protein; e.g., the binding of aspartate to ATCase –heterotropic effects: allosteric interactions that occur when different substances are bound to the protein; e.g., inhibition of ATCase by CTP and activation by ATP –Allosteric Activation –Allosteric Inhibition Heteroallostery The Concerted Model(WMC Model) • Wyman, Monod, and Changeux – 1965 WMC Model explains the sigmoidal effects of Heteroallostery •The enzyme has two conformations –R (relaxed): binds substrate tightly; the active form –T (tight or taut): binds substrate less tightly; the inactive form –in the absence of substrate, most enzyme molecules are in the T (inactive) form –the presence of substrate shifts the equilibrium from the T (inactive) form to the R (active) form Concerted Model –in changing from T to R and vice versa, all subunits change conformation simultaneously; all changes are concerted –an allosteric activator (A) binds to and stabilizes the R (active) form –an allosteric inhibitor (I) binds to and stabilizes the T (inactive) form Figure: Effect of binding activators and inhibitors Sequential Model •Koshland - 1966 –the binding of substrate induces a conformational change from the T form to the R form –the change in conformation is induced by the fit of the substrate to the enzyme, as per the induced-fit model of substrate binding Sequential Model Figure (a) Sequential model for cooperative binding of substrate to an allosteric enzyme Figure (b) Allosteric activation and inhibition also occur by the induced- fit mechanism
  44. 44. Two types of allosteric enzymes •For an allosteric enzyme, the substrate concentration at one-half Vmax is called the K0.5 –K system: an enzyme for which an inhibitor or activators alters K0.5 –V system: an enzyme for which an inhibitor or activator alters Vmax but not K0.5 Allosteric switch Covalent Modifications •There are many enzymes in cells which modify other enzymes: •phosphorylation (by protein kinases, PK) or dephosphorylation (by phosphoprotein phosphatase, PP) of various amino acid side chains (e.g., serine, threonine, tyrosine, and histidine). •proteolytic cleavage(by proteases), e.g. activation of zymogens Kinase cascade •A enzymatic cascade that leads to activation or inactivation of the last enzyme in the cascade by sequential phosphorylation. •Kinase cascade is mediated by hormone-receptor complex •Synthesis and degradation of Glycogen: A hormone-activated kinase cascade leads activation of glycogen synthase(GS) or glycogen phosphorylase (GP) Reproduction of yeast Overview of five MAP kinase pathways in S. cerevisiae Kinase cascade in the mating pathway in S. cerevisiae Structures of MAP kinase Coenzymes, Vitamins, and Essential Metals Coenzymes •In such cases, a protein may require the help of some other small molecule or ion to carry out the reaction. •Molecules bound to enzymes for this purpose are called coenzymes. •Many of the coenzymes are closely related to vitamins. Several important coenzymes 1. Nicotinamide adenine dinucleotide (NAD+ and NADH)
  45. 45. NAD + in some dehydrogenases • Ethanol + NAD+ <=> Acetaldehyde + NADH (Alcohol Dehydrogenase) NAD + in UDP-galactose epimerase NADP+ and NADPH •e.g CDP + NADPH <=> dCDP + NADP+ (ribonucleotide reductase) 2. FAD •Succinate + FAD (enzyme bound) <=> Fumarate + FADH2 (enzyme bound) FADH2 3. Thiamine Pyrophosphate (TPP) •pyruvate decarboxylase, •pyruvate dehydrogenase, •branched chain α-keto acid dehydrogenase, α-keto glutarate dehydrogenase, transketolase. 4. Coenzyme A (CoA or CoASH) Conenzymes and vitamins
  46. 46. Chapter 7 Hormone and Cell Signaling The word hormone  The word hormone is derived from the Greek verb horman, meaning "to stir up or excite."  Ernest Henry Starling introduced the term hormone in 1905 in a famous lecture "The Chemical Correlation of the Functions of the Body."  Hormones: Communication among Cells and Tissues  Endocrine cells secrete hormones  hormones are carried rapidly in the blood between distant organs and tissues; they may travel a meter or more before encountering their target cell (Fig.). Three classes of hormones:  There are three chemically distinct classes of hormones: peptides, amines, and steroids:  The peptide hormones, which may have from 3 to over 200 amino acid residues, include all of the hormones of the hypothalamus and pituitary and the pancreatic hormones insulin, glucagon, and somatostatin  The amine hormones, low molecular weight compounds derived from the amino acid tyrosine, include water-soluble epinephrine and norepinephrine of the adrenal medulla and the less watersoluble thyroid hormones  The steroid hormones, which are fatsoluble, include the adrenal cortical hormones, hormone forms of vitamin D, and the androgens and estrogens  A fourth group of extracellular signals, the eicosanoids, are hormonelike in their actions, but act locally. Concentration of hormones Hormones normally occur in very low concentrations in the blood, in the micromolar (10-6 M) to picomolar (10-12 M) range; Receptors of hormones  All hormones act through specific receptors present in hormonesensitive target cells, to which hormones bind with high specificity and high affmity. Each cell type has its own combination of hormone receptors, defining the range of its hormone responsiveness.
  47. 47.  The water-soluble peptide and amine hormones do not penetrate cell membranes readily; their receptors are located on the outer surface of the target cell  The lipid-soluble steroid and thyroid hormones readily pass through the plasma membrane of their target cells; their receptors are specific proteins located in the nucleus. second messenger  Upon hormone binding to a plasma membrane receptor, the receptor protein undergoes a conformational change analogous to that produced in an allosteric enzyme by effector binding. In its altered form, the receptor either produces or causes production of an intracellular messenger molecule, often called the second messenger.  Some second messengers: adenosine 3',5'-cyclic monophosphate (cAMP),,IP3, DAG, cGMP, Ca2+ Molecular Mechanisms of Signal Transduction Receptors for Epinephrine Trigger Cyclic AMP Production. (1) a hormone receptor in the plasma membrane; (2) the enzyme adenylate cyclase, which catalyzes cAMP formation; (3) Gs protein, which shuttles between the receptor and adenylate cyclase, activating the cyclase when hormone is bound to the receptor; (4) a cAMP-dependent protein kinase, which phosphorylates target enzymes within the cell, altering their activities; and (5) cyclic nucleotide phosphodiesterase, which degrades cAMP and thereby terminates the intracellular signal.  Adrenergic receptors: The four types (α1, α2, β1,β2) are found in different target tissues and mediate different responses to epinephrine.  β-adrenergic receptors –  in muscle, liver, and adipose tissue.  are integral membrane proteins with amino acid sequences that contain seven hydrophobic regions of 20 to 28 residues.  GTP-Bindtng Protein and Adenylate Cyclase:  located on the cytosolic face of the plasma membrane.  Gs stimulates the production of cAMP by adenylate cyclase.  Gs is composed of three polypeptides, α, β, and γ.  With GTP bound to the site, Gs is active, but with GDP bound to the site, Gs is inactive.
  48. 48.  Binding of epinephrine causes the receptor to catalyze the displacement of the GDP bound to inactive Gs by GTP; As this occurs, the β and γ subunits dissociate from the a subunit; Gsα;, with its bound GTP, then moves in the plane of the membrane from the receptor to a nearby molecule of adenylate cyclase.  Activation of adenylate cyclase by Gsα is self limiting;  Gsα has a weak GTPase activity and turns itself off by converting its bound GTP to GDP.  Cyclic AMP:  is short-lived; it is quickly degraded by cyclic nucleotide phosphodiesterase to 5'-AMP  Methyl xanthines such as theophylline (a component of tea) inhibit the phosphodiesterase, potentiating the action of agents that act through adenylate cyclase.  Cyclic AMP-Dependent Protein Kinase  Cyclic AMP does not affect phosphorylase b kinase directly. Rather, cAMP-dependent protein kinase, also called protein kinase A, which is allosterically activated by cAMP, catalyzes the phosphorylation of inactive phosphorylase b kinase to yield the active form.  The inactive form of cAMP-dependent protein kinase contains two catalytic subunits (C) and two regulatory subunits (R)  cAMP binds to two sites on each of the two R subunits, the R subunits undergo a conformational change, and the R2C2 complex dissociates to yield two free, catalytically active C subunits.  The Insulin Receptor  Is a Tyrosine-Specific Protein Kinase, which transfers a phosphate group from ATP to the hydroxyl group of Tyr residues.  The insulin receptor has two identical a chains that protrude from the outer face of the plasma membrane, and two transmembrane β subunits, with their carboxyl termini on the cytosolic face  The a chains contain the insulin-binding domain, and the β chains have the tyrosine kinase domain.  Insulin binding to the a chains activates the tyrosine kinase activity of the β chains. The enzyme first phosphorylates itself on critical Tyr residues in the β chain , this autophosphorylation activates the enzyme to phosphorylate other proteins of the membrane or cytosol.
  49. 49.  A third class of signal receptors and diacylglycerol and inositol-1,4,5 -trisphosphate  The signal receptors are coupled, through a G protein, to a plasma membrane phospholipase C specific for the plasma membrane lipid phosphatidylinositol-4,5-bisphosphate. This hormone-sensitive enzyme catalyzes the formation of two potent second messengers: diacylglycerol and inositol-1,4,5-trisphosphate. 2+  diacylglycerol is activating a membrane-bound, Ca -dependent enzyme, protein kinase C.  inositol-1,4,5-trisphosphate, the water-soluble product derived from phospholipase C action, diffuses from the plasma membrane to the endoplasmic reticulum, where it binds to specific receptors and causes Ca2+ channels within the reticulum to open, releasing sequestered Ca2+ into the cytosol.  Calcium Is a Second Messenger in Many Signal Transductions  Normally the cytosolic [Ca2+] is kept very low (<10-7 M) by the action of Ca2+ pumps in the endoplasmic reticulum, mitochondria, and plasma membrane.  Ca2+-dependent enzymes: Ca2+/calmodulin-dependent protein kinase  Calmodulin (Mr. 17,000) is an acidic protein with four high-affinity Ca2+-binding sites. When intracellular [Ca2+] rises to about 10-6 M (1μM), the binding of Ca2+ drives a conformational change in calmodulin.
  50. 50. Part 2 Metabolism Chapter 8 Overview of Metabolism Things to learn from this lecture What is metabolism The functions of metabolism Catabolic, anabolic pathways Characteristics of metabolic pathways Control of metabolic pathways An Overview of Metabolism Metabolism (change in Greek): the sum of the chemical changes that convert nutrients into energy and the complex finished products of cells. basic functions of metabolism 1. To obtain energy from fuel molecules or sunlight 2. To convert nutrients into precursors of cellular components. 3. To assemble precursors utilizing energy into cellular components. Metabolism Metabolism consists of catabolism and anabolism Catabolism: degradative pathways –Usually energy-yielding! Anabolism: biosynthetic pathways –energy-requiring! ANABOLIC CATABOLIC Chemical energy ATP - energy currency. It serves as the driving force for nearly all biochemical processes NADH,NADPH - reducing power. NADH for oxidation and energy yielding. NADPH for biosynthetic processes Catabolism( Degradation) Metabolic Pathways Characteristics of Metabolic Pathways Metabolic pathways are irreversible
  51. 51. Metabolic pathways have committed step Metabolic pathways are regulated Metabolic pathways are compartmentalized ( in eukaryotic cell) Control of metabolic pathways Molecular level Control of enzyme levels gene expression (slow) Control of enzyme activity (fast) allosteric control (binding of an effector at one site affects enzyme activity at another site) covalent control (phosphorylation, adenylylation, etc) Cellular level Compartmentation Mitochondria (TCA cycle, OxPhos, fatty acid oxidation, amino acid breakdown) Cytosol (glycolysis, fatty acid biosynthesis, pentose phosphate pathway) Nucleus (DNA replication, transcription, RNA processing) • Hormonal regulation Experimental Methods Inhibitors (accumulation of intermediates) Genetic defects (also accumulation of intermediates) Isotopic tracers Emerging methods: sequence methods (genome projects), bioinformatics, high throughput gene expression methods, (microarray analyses - hybridizations on membranes, slides, chips) Bioenergetics Gibbs Free Energy ∆G < 0 A reaction can occur spontaneously ∆G = 0 A system is at equilibrium: no net change occurs ∆G > 0 A reaction cannot occur spontaneously. An input of free energy is required to drive the reaction ∆G with a Small Magnitude usually means the reaction is reversible. ∆G with a Large Negative Magnitude Usually means the reaction is most likely irreversible.
  52. 52. ∆Go standard free energy change T = 298 K, concentration of all reactants is 1.0 M R = 1.98 cal/(mol*deg) Equilibrium Constants At equilibrium, t G°’ = 0, so Write the Keq for this reaction: Determination of Gº' DHAP ⇔ Glyceraldehyde-3-phosphate At equilibrium, [G-3-P]/[DHAP] = 0.0475.  ∆Gº' = -RTln(0.0475) = -1.98 x 10-3 kcal/(mol-deg) * 298 * (-3.047) = + 1.8 kcal/mol ∆G Values are Additive An example in the reaction catalyzed by Hexokinase, the half-reactions are: Pi + glucose  glucose-6-P + H2O e Go' = +14 kJ/mol ATP + H2O  ADP + Pi Go' = 31 kJ/mol Coupled reaction: ATP + glucose  ADP + glucose-6-P Go' = 17 kJ/mol Factors that Contribute to a Large Negative ∆G°' Types of Bonds in ATP major roles of ATP Energy tranducing mechanism- coupling all metabolic processes and therefore central to metabolic control Regulates a large number of enzyme reactions (ATP is an allosteric effector of many enzymes) Central Role of ATP in Metabolism Energy Charge (EC) Relationship between the EC and Metabolism Overview of metabolism and Bioenergetics •Three stages of catabolism (respiration) •Gibbs Free Energy Glycolysis
  53. 53. Pathway Energetic Regulation Cellular function /Location Citric Acid Cycle Pathway Energetic Regulation Cellular function / Location Pentose Phosphate Pathway •Metabolic Significance Electron transport chain Electron transport chain Chemiosmotic hypothesis Binding change mechanism Drugs that inhibit electron transport and oxidative phosphorylation Location Gluconeogenesis •Precursors for Gluconeogenesis •Gluconeogenesis and glycolysis are reciprocally regulated Fatty Acid β-Oxidation •Location •Pathway •Energetics Fatty acid synthesis •Intracellular location •Pathway (Building blocks, Reducing power,Acyl carriers) Degradation of amino acids •Deamination •Urea cycle DNA Replication •Features of DNA Replication •How to ensure the accuracy of DNA Replication •Proteins in DNA replication (DNA polymerase, primase, SSB , helicase, Gyrase ,Ligase) •Steps of DNA Replication
  54. 54. Transcription •Differences between DNA and RNA polymerases •E. coli RNA polymerase subunits and their function •Promoters •lac Operon •Post-transcriptional processing of mRNA in Eukaryotes Translation •Shine-Dalgarno (S-D) sequence •How to ensure the accuracy of translation •Process of protein synthesis •If you add pyruvate labeled in the carboxylate group to actively metabolizing eukaryotic cells under aerobic conditions, under what conditions will that labeled carbon enter the citric acid cycle? •Lactate is a good substrate for hepatic fatty acid synthesis. Give the pathway by which metabolism of lactate in the liver can give rise to the active two-carbon unit and the reducing power required for fatty acid synthesis. Identify each of the following pathways or processes and indicate the cellular location 1 Synthesizes ATP from ADP driven by the H+ gradient across the inner mitochondrial membrane. 2 Transfers electrons ultimately to O2 in a series of redox reactions that produces across H+ gradient the inner mitochondrial membrane. 3 Chops 2 carbons off a fatty acid each time it goes around the cycle. Each 2 carbon unit becomes acetyl CoA. 4 Accepts 2 carbons from acetyl CoA which combine with oxaloacetic acid to form citric acid. Generates CO2 and reduced coenzymes (NADH and FADH2). 5 Converts 1 mole of glucose to 2 moles of pyruvate
  55. 55. Chapter 9 Energy Metabolism 9.1 glycolysis-Anaerobic Metabolism of glucose Things to Learn •Pathway •Energetics •Regulation •Cellular function / localization An overview on glucose metabolism •The major fuel of most organisms, releasing large energy if completely oxidized to CO2 and H2O via the glycolysis (糖酵解), citric acid cycle(柠 檬酸循环) and oxidative phosphorylation (氧化磷酸化) . •Can also be oxidized to make NADPH and ribose-5-P via the pentose phosphate pathway(磷酸戊糖途径). •Can be stored in polymer form (glycogen or starch) or be converted to fat for long term storage. •Is also a versatile precursor for carbon skeletons of almost all kinds of biomolecules, including amino acids, nucleotides, fatty acids, coenzymes and other metabolic intermediates. Glycolysis •Glycolysis:from the Greek glyk-sweet, lysis-splitting •--the stepwise degradation of glucose, the conversion of glucose into pyruvate(丙酮酸) •Glycolysis means the anaerobic metabolism of glucose 1. The Development of Glycolysis •1897, Eduard Buchner (Germany), accidental observation : sucrose (as a preservative) was rapidly fermented into alcohol by cell-free yeast extract. (1907 Nobel Prize laureate) •Metabolism became chemistry! •1900s, Arthur Harden and William Young (Great Britain)separated the yeast juice into two fractions: one heat-labile, nondialyzable zymase (enzymes) and the other heat-stable, dialyzable cozymase (metal ions, ATP, ADP, NAD+). (1929 Nobel Prize laureate) •1910s-1930s, Gustav Embden and Otto Meyerhof (Germany), studied muscle and its extracts:
  56. 56. –Reconstructed all the transformation steps from glycogen to lactic acid in vitro; revealed that many reactions of lactic acid (muscle) and alcohol (yeast) fermentations were the same! –Glycolysis was also known as Embden-Meyerhof pathway. ( 1922 Nobel Prize laureate) •The whole pathway of glycolysis (Glucose to pyruvate) was elucidated by the 1940s. 2. The overall glycolysis pathway can be divided into two phases •Ten steps of reactions are involved in the pathway. •The first 5 reactions are called the preparatory phase of glycolysis. The hexose is first activated and then cleaved to two three-carbon intermediates, consuming ATP. •The remaining reactions are called the payoff phase of glycolysis. The three-carbon intermediates are then oxidized, generating ATP and NADH. •All intermediates are phosphorylated (as esters or anhydrides). •Only a small fraction (~5%) of the potential energy of the glucose molecule is released and much still remain in the final product of glycolysis, pyruvate. •All the enzymes are found in the cytosol. 3. Ten enzymes catalyze the ten reactions of glycolysis The preparatory Phase of glycolysis ATP binds to the enzyme as a complex with Mg++. •The first priming reaction •Traps glucose inside cells •Irreversible 2. Phosphoglucose Isomerase(磷酸葡萄糖异构酶) The payoff phase of glycolysis
  57. 57. The “high energy” acyl thioester is attacked by Pi to yield the acyl phosphate (~P) product. This step can be bypassed by Arsenate(砷酸盐,analogous to phosphate). total reaction: glucose + 2 NAD+ + 2 ADP + 2 Pi  2 pyruvate + 2 NADH + 2 ATP+2H++2H2O 4 Fate of Pyruvate and Regeneration of NAD+ Why should NAD+ be recycled? •NAD+ is reduced to NADH during glycolysis •The amount of NAD+ in the cell is small •In order for glycolysis to continue, the NADH formed must be reoxidised to NAD+ Regeneration of NAD+ •Aerobic condition. NAD+ is recycled by electron transfer chain and 2 or 3 ATP are synthesized by oxphos(氧化磷酸化). •Anaerobic condition. Hydrogen of NADH is transferred to the pyruvate. Ethanol Fermentation Some anaerobic organisms metabolize pyruvate to ethanol, The above pathway regenerates NAD+, needed for continuation of Glycolysis. Total reaction: C6H12O6+2ADP+2Pi→ 2C2H6O+2CO2+2ATP+2H2O 5 Energetics of glycolysis How many ATP bonds expended? How many ATP produced in the pathway? (Remember there are two 3C fragments from glucose.) Net production of ATP per glucose in Anaerobic and aerobic conditions. •2ATP expended •4ATP produced through substrate level phosphorylation Net production of ATP •In anaerobic condition, 2ATP •In aerobic condition, 6-8ATP ( when NADH is oxidized through electron transfer chain , 2 or 3 ATP are synthesized by oxphos) 6 Regulation of Glycolysis
  58. 58. •a pathway is controlled at rate-limiting steps •Flux through the rate-determining steps may be altered by several mechanisms –1. Allosteric control –2. Covalent modifications Regulation of the enzymes of Glycolysis 3 irreversible reactions •Hexokinase –HK is allosterically inhibited by the product, G6P •Pyruvate Kinase –ATP and alanine are allosteric inhibitors –F-1,6-2P is an allosteric activator •Feed forward activation –Inactivated by phosphorylation as a result of the activation of cAMP- dependent protein kinase (PKA) via Glucagon ( 胰高血糖素) •Most key rate-limiting step of Glycolysis •Allosterically inhibited by ATP, citrate Inhibition by ATP is relieved by AMP Energy charge is an index of cellular energy status E.C. = (ATP+1/2ADP) / (ATP+ADP+AMP) •The most potent allosteric activator of PFK-1 in liver is fructose-2,6- bisphosphate (F-2,6-P) 7. Entry of other sugars into the glycolytic pathways •Other hexoses are also oxidized via the glycolysis •They are also first primed by phosphorylation (at C-1 or C-6). 8. Dietary poly- and disaccharides are hydrolyzed to monosaccharides in the digestive system •-amylase- Salivary -S (淀粉酶) in the mouth hydrolyzes starch into short polysaccharides or oligosacchrides. •-amylase- Pancreatic (active at low pH) continue act to convert the saccharides to mainly maltoses and dextrins (from amylopectin, 枝链淀粉). •Specific enzymes (e.g., lactase, sucrase, maltase,etc.) on the microvilli of the intestinal epithelial cells finally hydrolyze all disaccharides into monosaccharides. •The monosacchrides are then absorbed at the intestinal microvilli and
  59. 59. transported to various tissues for oxidative degradation via the glycolytic pathway. 9 Glycogen Breakdown •Glycogen –1-4 glucose1 A high molecular weight glucose polysacharide comprised of 1-6 linkages(at branches )o linkages (mainly) and –Found mainly in Muscle (1-2% by weight) and Liver (up to 10% by weight) The Structure of Glycogen LIVER MUSCLES •When blood glucose drops below normal (2-3 hours after the last meal) •Maintains blood glucose •Continues until: –Next meal or –Liver glycogen is depleated (12-24 hours) •To provide energy for muscles during strenuous exercise (i.e. when anaerobic conditions prevail) •Does NOT contribute significantly to blood glucose Glycogenolysis •Glycogen Phosphorylase •By phosphorolysis: Splits bonds by incorporating Pi •Continues until there are 4 glucose units on each side of the branch Debranching Enzyme Glycogenolysis •G1P produced in the Glycogen breakdown is converted to G6P in a reaction catalyzed by Phosphoglucomutase •G6P then has different fates in different tissues Regulation of Glycogen breakdown •Glycogen Phosphorylase is allosterically activated by AMP and inhibited by ATP, glucose-6-P •Glycogen Phosphorylase is regulated by covalent modification - phosphorylation Covalent modification •Glucagon & epinephrine activate cAMP cascades •The cAMP cascade results in phosphorylation of a serine hydroxyl of Glycogen Phosphorylase
  60. 60. •phosphorylation promotes transition of b (less active ) state to the a(active) state. •The cAMP cascade activates glycogen degradation. Home work •The cell uses many strategies to drive an energetically unfavorable reaction forward. Identify two such strategies and give an example from glycolysis of a reaction that demonstrates one of these strategies. 9.2 Aerobic Metabolism of carbohydrate The three stages of respiration •Stage I All the fuel molecules are oxidized to acetyl-CoA. •Stage II The acetyl-CoA is completely oxidized into CO2, electrons were collected by NAD and FAD via the citric acid cycle. •Stage III Passage of electrons through the electron transport system to yield ATP from oxidative phosphorylation. Pyruvate Oxidation Pyruvate + CoA + NAD+  acetylCoA + CO2 + NADH + H+ Pyruvate Dehydrogenase is a large complex: pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), dihydrolipoyl dehydrogenase (E3) Requires 5 coenzymes: TPP, Lipoic Acid, Coenzyme A, FAD, NAD+ Reactions of the PDH complex Regulation of Pyruvate Dehydrogenase 1). Product inhibition by NADH & acetyl CoA 2). Covalent modification Citric Acid Cycle •Hans Krebs proposed the “citric acid cycle” for the complete oxidation of pyruvate in animal tissues in 1937 (1953 Nobel Prize laureate). •The tricaboxylic acid (TCA) Cycle, Krebs Cycle Citric Acid Cycle •The common pathway leading to complete oxidation of carbohydrates, fatty acids, and amino acids to CO2. •Some ATP is produced, More NADH is made ,NADH goes on to make
  61. 61. more ATP in electron transport and oxidative phosphorylation •A pathway providing many precursors for biosynthesis citric acid cycle overview Individual reaction Reaction 1: Citrate Synthase Fluoroacetate blocks the cycle •Fluoroacetate is poisonous because it can convert to fluorocitrate which is an inhibitors of TCA cycle. Reaction 2: Aconitase Reaction 3: Isocitrate →Dehydrogenase Reaction 4: α-Ketoglutarate → Dehydrogenase Reaction 5: Succinate thiokinase Reaction 6: Succinate Dehydrogenase Succinate Dehydrogenase •Part of electron transport chain in the inner membrane of mitochondria. •Removal of H across a C-C bond is not sufficiently exergonic to reduce NAD+,but it does yield enough energy to reduce FAD. •Malonate is a competitive inhibitor Reaction 7: Fumarase Reaction 8: Malate Dehydrogenase TCA Cycle Summary Aerobic Nature of the Cycle Energetics ATP generated by the cycle Glucose glycolysis 2ATP(Substrate-level phosphorylation) 2NADH ( oxphos) 2Pyruvate oxidative decarboxylation 2 NADH ( oxphos)
  62. 62. 2 Acetyl CoA TCAcycle 6 NADH 2 FADH2 2 GTP total 30-32ATP The Fate of Carbon in TCA Reaction 2: Aconitase •Carboxyl C of acetate turns to CO2 only in the second turn of the cycle •Methyl C of acetate survives two cycles completely, but half of what's left exits the cycle on each turn after that. Regulation of the TCA Cycle Again, 3 irreversible reactions are the key sites •Citrate synthase - regulated by availability of substrates - acetyl-CoA and oxaloacetate, citrate is a competitive inhibitor; Allosteric: - NADH , ATP,succinyl-CoA •Isocitrate dehydrogenase – NADH,ATP inhibit, ADP and NAD+ Ca++ activate • -Ketoglutarate dehydrogenase - NADH and succinyl-CoA inhibit, AMP Ca++activate Anaplerotic reactions •Anaplerotic (filling up) reactions replenish citric acid cycle intermediates •Amphibolic Nature of TCA Cycle means it both Anabolic and Catabolic. TCA cycle provides several of Intermediates for Biosynthesis Anaplerotic reactions •PEP carboxylase - converts PEP to oxaloacetate , Anaplerotic reaction in plants and bacteria •Pyruvate carboxylase - converts pyruvate to oxaloacetate, a major anaplerotic reaction in mammalian tissues •Malic enzyme converts pyruvate into malate The Glyoxylate Cycle An Anabolic Variant of the Citric Acid Cycle for plants and bacteria
  63. 63. •Acetate-based growth - net synthesis of carbohydrates and other intermediates from acetate - is not possible with TCA •Glyoxylate cycle offers a solution for plants and some bacteria and algae •The CO2-evolving steps are bypassed and an extra acetate is utilized •Isocitrate lyase and malate synthase are the short-circuiting enzymes Home work •Starting with pyruvate labelled with 13C on the keto-carbon, where will this isotopic label end up in oxaloacetate after one round of the citric acid cycle? Briefly describe •The reaction catalyzed by the pyruvate dehydrogenase complex in carbohydrate metabolism: •One advantage of a multienzyme complex •explain how pyruvate dehydrogenase is regulated. (Graphs of enzyme activity would be cool)! •What effect would increasing expression of the PDH kinase gene have on carbohydrate metabolism? •If you add pyruvate labeled in the carboxylate group to actively metabolizing eukaryotic cells under aerobic conditions, under what conditions will that labeled carbon enter the citric acid cycle? •What is the primary regulatory site in glycolysis? Explain the role of energy charge in controlling the enzyme. •Name other sites for regulation in glycolysis. 9.3 Lipid metabolism Digestion of dietary lipids •There are 2 important secretions that are essential for digestion of triglycerides: 1) Bile salts Act as emulsifier 2) Pancreatic lipase Removes 2 fatty acids from triglycerides Action of pancreatic lipase
  64. 64. Triacylglycerol degradation Why Fatty Acids are used for storage of energy? Two reasons: –The carbon in fatty acids (mostly CH2) is almost completely reduced (so its oxidation yields the most energy possible). –Fatty acids are not hydrated (as mono- and polysaccharides are), so they can pack more closely in storage tissues Result: fatty acids have ~6 more energy of the corresponding amount of proteins or glycogen Hormones signal the release of fatty acids from adipose tissue Fate of Glycerol –From action of lipoprotein lipase and hormone-sensitive lipase –Glycerol is converted to the Glycolysis/ gluconeogenesis intermediate-- dihydroxyacetone phosphate Glycerol is converted to dihydroxyacetone phosphate, by reactions catalyzed by: 1 Glycerol Kinase 2 Glycerol Phosphate Dehydrogenase. -Oxidation of fatty acids- Martius & Knoop (1902) fed dogs even- and odd-carbon fatty acids labelled with a benzene ring in place of the terminal methyl group. •Fatty acid -carbonF oxidation occurs by removal of 2-C units at a time with oxidation at the of the fatty acid Fatty Acid β-Oxidation •All cells except for RBCs and brain can use fatty acids for energy. •β-Oxidation occurs in Mitochondria •Three Steps –A.activation –B.transport into mitochondria –C.oxidation A. Fatty acid activation Acyl-CoA Synthetase of ER & outer mitochondrial membranes, catalyzes fatty acid activation. Fatty Acid Activation fatty acid + ATP  acyladenylate + PPi

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