Bioenergentics/Enzymes 2016

10/24/1610/24/16 15:0815:08 cottinghamcottingham
Bioenergetics:Bioenergetics:
Protein & EnzymesProtein & Enzymes

10/24/16 15:08 cottingham
Classes of Macromolecules in Cells
Carbohydrates
Lipids
Proteins
Nucleic Acids

 Proteins either achieve all cell
structure and function OR they
make all of the molecules and
structures which gives function
to cells.

 Set of expressed proteins
in a particular cell or
organism under a given
set of environmental
conditions.

Proteins
• Contain Carbon, Hydrogen, Oxygen,
Nitrogen…sometimes Sulfur.
•Building blocks (monomers) are
Amino Acids.
•Polymers (macro) are polypeptide
chains: 50 to 30,000 amino acids
long

 Structural
 Components of all cell membranes
 Component of cytoplasm
 “ “of movement
 “ “ of hair, nails, horns, etc
 Metabolic
 Hormones regulatory chemical
 Energy transfer molecules for cell respiration
 Oxygen carrier in circulation
 Antibodies
 Enzymes ***

 Five parts – first 4 are the same in all a.a.
 Central carbon
 Hydrogen
 Amino group (-NH2)
 Carboxyl group (-COOH)
 R group
 What makes one amino acid different from another
 All proteins are polymers composed of the
same set of 20 possible amino acids

General Amino Acid Structure
There are 20
different
amino acids.
Each has a
different “R”
group.

 Depends on the “R”
group
 Nonpolar:
 Have non polar R
groups
 CH
 Tend to be
hydrophobic
 Polar:
 Have polar R groups
 Hydroxyl or ketone
 Tend to be
hydrophilic

R group

R
groups

•One group of amino acids has hydrophobic R
groups.

Condensation ReactionCondensation Reaction
 Forms dipeptidesForms dipeptides
 Removes hydroxyl group from carboxyl group of a.a.Removes hydroxyl group from carboxyl group of a.a.
 Removes hydrogen from amino group of a.aRemoves hydrogen from amino group of a.a
 Removes 1 waterRemoves 1 water
 The resulting covalent bond is called aThe resulting covalent bond is called a peptide bondpeptide bond..

C3032H4816O872N780S8Fe4

val his leu thr pro glu glu lys ser ala val thr ala leu tyr gly lys val
asn val asp glu val gly gly glu ala leu gly arg leu leu val val tyr pro
try thr gln arg phe phe glu ser phe gly asp leu ser thr pro asp ala val
met gly asn pro lys val lys ala his gly lys lys val leu gly ala phe ser
asp gly leu ala his leu asp asp leu lys gly thr phe ala thr leu ser gln
leu his cys asp lys leu his val asp pro glu asn phe arg leu leu gly asn
val leu val cys val leu ala his his phe gly lys glu phe thr pro pro val
gln ala ala tyr gln lys val val ala gly val ala asp ala leu ala his lys tyr
his

Levels of Protein Structure
FOLDING OF PROTEINS

 The simplest sequence of amino acids
 Sequence plays the biggest role in the shape
that a protein takes on
 Primary structure is unique for every kind of
polypeptide
 Even a tiny change in the primary structure
can have a profound effect on the function of
a protein
 PEPTIDE BONDS

 Folding that is a result of hydrogen bonding between
a.a.
 Two major secondary structures
 Alpha helix
 Coil held together by H bonding between every
4th
a.a
 keratin – hair and skin
 Beta pleated sheet
 2 or more regions lie parallel and H bond together
 Silk

 The overall 3 dimensional structure that a protein has.
 Folding of a poly peptide due to the type of bond:
 Peptide Bond – Primary bonding
 Hydrogen bonding – between polar “R” groups
 Ionic bonding – electrovalent bonding – charged “R”
 Disulfide Bridges - 2 sulfur atoms bond using any a.a. that
contains sulfur: cysteine (Covalent Bond)
 Vander Waals Forces – between non-polar a.a.

 Must be made up of more than one
polypeptide
 Hemoglobin (4) ; many enzymes
 Quaternary proteins may form CONJUGATED
PROTEINS
 Protein containing non protein parts
 Non protein part: prosthetic group

Fibrous
 Proteins in 2ndary
structure; repeated
sequences of a.a. in long
sheet
 Water insoluble
 Very tough
Structural role in cells
 Muscle proteins**
 Keratin – hair
 Collagen
Globular
 Polypeptide chains in the
3° or 4°; folded into
rounded shape
 Water soluble
 Critical to function of the
body:
 Catalytic – enzymes
 Regulatory – hormones
 Transport – hemoglobin
 Immune – antibodies
 Structure - microtubule

• Loss of the 3-dimensional structure and function of a
protein. (Break Down)
• May be permanent
• Results from an alteration of bonds that maintain the
2ndary and 3rdary structure
• Caused By:
• Strong acids and bases
• Heavy Metals
• Heat and Radiation(UV)
• Detergents and solvents

 Enzymes
 Catalyze reactions – Catalase, RUBISCO
 Structural functions
 KERATIN - alpha
 COLLAGEN - alpha
 Major structural protein of the skin
 SPIDER SILK - beta
 Sight – RHODOPSIN – found in Rod cells
 Contractile – muscle contraction
 Actin and myosin
 Transport
 Help control what goes in/out of cells
 Hemoglobin – carries oxygen from lungs throughout
body in blood

 Hormones
 Signal molecules in the body
 HGH – it’s a protein!!
 INSULIN-regulates sugar concentrations
 Receptors
 Help cells recognize molecules
 On cell membranes
 Immune system
 Antibody proteins in the immune system help
fight disease
 IMMUNOGLOBULINS

Bioenergetics

Metabolic ReactionsMetabolic Reactions
 Metabolism – the “web” of all the enzymeMetabolism – the “web” of all the enzyme
catalyzed reactions in a cell or organismcatalyzed reactions in a cell or organism
Anabolism – synthesis of complex moleculesAnabolism – synthesis of complex molecules
from simpler moleculesfrom simpler molecules
Catabolism – breakdown of complexCatabolism – breakdown of complex
molecules into simpler moleculesmolecules into simpler molecules

• Act asAct as catalystscatalysts for metabolic reactionsfor metabolic reactions
• AreAre globular proteinsglobular proteins with specific 3Dwith specific 3D
formationsformations
• Have aHave a high specificityhigh specificity forfor substratessubstrates
• SubstrateSubstrate – the molecule on which the– the molecule on which the
enzyme acts.enzyme acts.
• Possess an “Possess an “active siteactive site” on the surface of” on the surface of
the enzyme in which substrates readilythe enzyme in which substrates readily
interactinteract
How Enzymes Work
How Enzymes Work

Enzymes
 Enzymes are the catalysts of metabolic
rxns.
 Catalyst – affects a chemical reaction without
itself being changed (re-used).
 Most enzymes end in “ase” .
FUNCTION: Enzymes
regulate the rate at which
reactions occur.

Mechanisms of Enzyme ActionMechanisms of Enzyme Action

Lock & Key ModelLock & Key Model
 Enzyme –Enzyme –
SubstrateSubstrate
Specificity!Specificity!
Substrate
Enzyme
Active Site

 Usually large globularUsually large globular
proteins (a)proteins (a)
 b)b) Active SiteActive Site where thewhere the
substrate combines to thesubstrate combines to the
enzymeenzyme
 c) Substrate which fits thec) Substrate which fits the
active siteactive site
 d)d) enzyme-substrateenzyme-substrate
complexcomplex. The substrate is. The substrate is
weakened to allow theweakened to allow the
reaction.reaction.
 e) Unchanged enzyme/ re-e) Unchanged enzyme/ re-
used at low concentrationsused at low concentrations
 f)f) Product of the reactionProduct of the reaction
•Active site may have polar amino acids on the outside.
•Enzyme specificity is due to the “complementary” shape of the
active site and substrate.
•Enzymes work at low concentrations.

TheThe induced fitinduced fit between an enzyme and itsbetween an enzyme and its
substratesubstrate

 Active site is not a rigid pocket for the substrate toActive site is not a rigid pocket for the substrate to
fit in.fit in.
 Substrate “induces” the enzyme to change shapeSubstrate “induces” the enzyme to change shape
 Weakens the bonds of the substrateWeakens the bonds of the substrate
 Lowers the activation energyLowers the activation energy

The catalytic cycle of an enzymeThe catalytic cycle of an enzyme

InhibitorsInhibitors
General function:General function:
 Reduce activity of enzymeReduce activity of enzyme
 Stop enzyme completelyStop enzyme completely
2 General types:2 General types:
CompetitiveCompetitive
Non CompetitiveNon Competitive
IrreversibleIrreversible
Allosteric – includes feedback inhibitionAllosteric – includes feedback inhibition

Metabolic PathwaysMetabolic Pathways
Metabolic PathwaysMetabolic Pathways

Competitive InhibitorCompetitive Inhibitor
 Very similar to the substrateVery similar to the substrate
 Compete with the substrate for position`Compete with the substrate for position`
on the active siteon the active site
 ““binds” to the active sitebinds” to the active site
 NO reaction takes placeNO reaction takes place
 SLOW overall the rate of reactionSLOW overall the rate of reaction
 Prevents accumulation of substancesPrevents accumulation of substances

Competitive Inhibitor - ExampleCompetitive Inhibitor - Example
Krebs CycleKrebs Cycle
 Malonate – inhibitor of cell respirationMalonate – inhibitor of cell respiration
 Binds to the active site of the enzymeBinds to the active site of the enzyme
“Succinate dehydrogenase”“Succinate dehydrogenase”
 Interrupts Succinate to Fumarate step inInterrupts Succinate to Fumarate step in
the cyclethe cycle

Krebs's Cycle – A METABOLIC PATHWAY

Non - CompetitiveNon - Competitive
 Molecules NOT similar to the substrateMolecules NOT similar to the substrate
NO competition for the active siteNO competition for the active site
 ““Bind” to another site on the enzymeBind” to another site on the enzyme
 Inhibitor changes the conformation (3-D,Inhibitor changes the conformation (3-D,
tertiary structure) of the enzyme…tertiary structure) of the enzyme…
 causing enough alteration to slow enzymecausing enough alteration to slow enzyme
activity…activity…
 while substrate may bind to the activewhile substrate may bind to the active
site, it is not converted to a product….site, it is not converted to a product….

TWO TYPESTWO TYPES

Allosteric InhibitorsAllosteric Inhibitors
 Non competitive (end – product inhibition)Non competitive (end – product inhibition)
 Bind to a site in the enzyme:Bind to a site in the enzyme: allosteric siteallosteric site..
 RESULTS:RESULTS:
Might change active site so substrate can fitMight change active site so substrate can fit
 Increases reaction rateIncreases reaction rate
Might “distort” active site and inhibit reaction****Might “distort” active site and inhibit reaction****
 Lowers rate of reactionLowers rate of reaction
 Can control the rate of metabolic reactionsCan control the rate of metabolic reactions
ATP – feedback inhibitionATP – feedback inhibition

End-Product InhibitionEnd-Product Inhibition
using an Allosteric Inhibitorusing an Allosteric Inhibitor
 ““feedbackfeedback
inhibition”inhibition”
 ““End productEnd product
inhibition”: the lastinhibition”: the last
product of aproduct of a
reaction acting asreaction acting as
an inhibitor foran inhibitor for
another enzyme inanother enzyme in
the reaction.the reaction.
Feedback Inhibition

ExampleExample
ATP – Feedback inhibitionATP – Feedback inhibition (during glycolysis)(during glycolysis)
 ATP accumulatesATP accumulates
 Acts as an allosteric inhibitor for theActs as an allosteric inhibitor for the
enzyme “phosphofructokinase.” PFKenzyme “phosphofructokinase.” PFK
 Glucose --------Glucose -------- Fructose 1,6 BiphosphateFructose 1,6 Biphosphate
 Lowers the rate of reactionLowers the rate of reaction
 Less ATP is producedLess ATP is produced

Irreversible inhibitorsIrreversible inhibitors
 Generally are poisonous substances thatGenerally are poisonous substances that
enter from the outside of the body.enter from the outside of the body.
 Binds to the enzyme “irreversibly”Binds to the enzyme “irreversibly”
 Interferes with the binding of the enzymeInterferes with the binding of the enzyme
with the substrate…metabolic pathwaywith the substrate…metabolic pathway
stoppedstopped
 Examples:Examples:
 CN (cyanide gas) – inhibits “cytochrome oxidase” duringCN (cyanide gas) – inhibits “cytochrome oxidase” during
respirationrespiration
 Lead and other heavy metals: binds to “sulfur” in enzymesLead and other heavy metals: binds to “sulfur” in enzymes
 Nerve Gas – blocks an enzyme needed for the neurotransmitterNerve Gas – blocks an enzyme needed for the neurotransmitter
acetylcholineacetylcholine
 Antibiotics – penicillin – inhibits bacterial enzymes needed toAntibiotics – penicillin – inhibits bacterial enzymes needed to
build the Cell Wall – growth and reproduction STOPSbuild the Cell Wall – growth and reproduction STOPS

Inhibition AnimationsInhibition Animations
Ace inhibitorsAce inhibitors
The Science of Enzyme InhibitionThe Science of Enzyme Inhibition

Enzyme Action and EnergyEnzyme Action and Energy

How Do Enzymes Work?How Do Enzymes Work?
 For a reaction to start it needs energy:For a reaction to start it needs energy:
ACTIVATION ENERGYACTIVATION ENERGY
 Enzymes LOWER the Activation EnergyEnzymes LOWER the Activation Energy

Enzymes and Reactions
 Reactions are not impossible without
enzymes.
 With enzymes the RATE of reactions will
increase.
 Enzymes do no change the “contents” of the
reactions.

Energy in a Reaction
 Substrates must absorb energy from
their surroundings for their bonds to
break.
 Products release energy when their new
bonds are formed.
 The amount of energy needed for a
chemical reaction to proceed is the
ACTIVATION ENERGY, EA.

DRAW

Endergonic ReactionEndergonic Reaction
Endergonic ReactionEndergonic Reaction (Endothermic)(Endothermic) ––
Absorb energy into the reactionAbsorb energy into the reaction
Amount of energy in systemAmount of energy in system
increasesincreases

Endergonic Rxn.Endergonic Rxn.

Exergonic ReactionExergonic Reaction
Give off EnergyGive off Energy
Represented by a negativeRepresented by a negative
change in energychange in energy
Loss of free energyLoss of free energy

Energy profile of an exergonic reactionEnergy profile of an exergonic reaction

Figure 6.13 Enzymes lower the barrier of activation energyFigure 6.13 Enzymes lower the barrier of activation energy

Catalyzed ReactionsCatalyzed Reactions
The Small Intestine Reactions:The Small Intestine Reactions:
Starch +Starch + AmylaseAmylase = many maltose units= many maltose units
Maltose +Maltose + maltasemaltase = glucose + glucose= glucose + glucose
Lactose +Lactose + lactaselactase = glucose + galactose= glucose + galactose
Sucrose +Sucrose + sucrasesucrase = glucose + fructose= glucose + fructose
These are ALL hydrolytic reactions!!These are ALL hydrolytic reactions!!

Characteristics of Enzyme Activity
1. Enzymes work best at certain temperatures and
enable cell reactions to proceed at normal
temperatures.
2. Small amount of enzyme can affect large amounts
of substrate.
3. Enzyme and Substrate concentration can control
the rate of the reaction.
4. Enzymes work best at a certain pH.
5. A Co-enzyme may be needed for an enzyme to
function correctly.

Characteristics of Enzyme Activity
THE MAD GOOD ANNY’

Figure 6.16 Environmental factors affecting enzyme activityFigure 6.16 Environmental factors affecting enzyme activity

Temperature
 As you increase
temperature, enzyme
action increases as well
until an optimum
temperature for enzyme
action is reached.
 Often doubling with every
10°C rise
 “collisions” between
substrate and active site
more frequent at higher
temps.

Enzymes are temperature
dependent…... (in humans)
0
10
20
30
40
50
60
70
80
10
oC
20 37 40
 Most work at body
temperature:37o
C to
maintain homeostasis
 Denature at high
temperatures
 Inactive at low
temperatures

Effect of Substrate Concentration
•As substrate concentration
increases, the rate of enzyme
catalyzed reactions will
increase….and then become
constant.
• The increased
concentration is a “limiting
factor”
• When all active sites are
engaged by substrates,
reaction rate will not
increase.

pH
 Affects enzyme action.
 Certain enzymes work better in acidic
environments while others in basic
environments.

Effect of pH on Enzyme Activity
 Enzymes have
an optimum pH
at which they
function best.
 At other pH’s,
enzymes are
denatured,
because the H+
and OH-
ions
disrupt hydrogen
bonds that hold
the 3-D structure
in place.
Effect of pH on two different
enzymes: pepsin and
trypsin

Enzymes are pH specific.
 Different enzymes
 Different body areas
 Different optimum pH
 Examples:
Stomach= 1.5 to 4.0
Mouth = 6.5 – 7.5
Blood = normal – 7.4
Blood

Application of
Enzymes in
Biotechnology

Pectinase – ******IB Req.
 Pectin – polysaccharide found in
fruit skins (cell wall)
 Pectinase – found in natural
fungus (Aspergillus niger) on fruit.
Uses enzyme to soften fruit.
 USES: Hydrolyzes pectin into its
monomers.
 Pectinase is added during the
crushing of fruit
 ADV: Makes juice clearer &
increases volume! Easy to
separate from pulp.
Making Juice

Lactose Intolerance

Lactase
Source: Industrial lactase is produced
from fungus: Kluyveromyces lactis (K.
lactis)
Enzyme is “immobilized”
Added to milk. Concentration of milk
drops…glucose rises.
ADV:
1.Reduces intolerance
2.Sweeter
3.Use in ice cream…less crystallization
4.Uses in yogurt…bacteria ferment glucose faster
Can be expensive.

Biological Detergents
 Proteases – protein (most dirt)
 Source: Bacteria – Bacillus
licheniformis
 Contain enzymes to digest
stains at lower temperatures
than 45 C and high pH’s
 Wash in cold water!...lower
energy use, less shrinkage.

Co-existing with Enzymes:
Cofactors and
Coenzymes

COENZYMES

COENZYMES
 A type of cofactor ---- ORGANIC in nature
 Separate from the protein in the enzyme to react
DIRECTLY in the chemical reaction.
 Function: transfer electrons, atoms, or molecules
between enzymes
 Vitamins – help make coenzymes
 Vitamin B – helps to make NAD a coenzyme in cellular
respiration.
 NAD – electron carrier

Cofactors
 Needs to be present in addition to an
enzyme for a reaction to catalyze.
 Like enzymes, they return to their original
state when their reactions are completed.
 Inorganic – potassium, zinc, magnesium, iron
(not H2O)

Making Cheese
 Rennin – an enzyme that
turns milk to cheese.
 Coagulates into curd at
body temperature
 Found in stomachs of
babies – helps to retain
food
 Found in stomachs of
young cattle*****

Bioenergentics/Enzymes 2016

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