Globular proteins are characterized as generallyhaving:• a variety of different kinds of secondary structure• spherical shape• good water solubility• a catalytic/regulatory/transport role i.e. a dynamic metabolic function
Globular heme proteins contain heme as prostheticgroup.Functions of globular hemeproteins include: • electron carriers • part of enzyme active site • transport of O2 and CO2- hemoglobin • storage of O2-myoglobin
• II. Globular Hemeproteins • Contain heme as prosthe.c group • Role of heme is dependent on environment created by 3D structure of protein • Heme of cytochrome → electron carrier • Heme of catalase → part of ac.ve site • Heme of Hb and myoglobin → binds O2 reversibly
• A. Structure of Heme • Complex of Protoporphyrin IX & Fe2+ • Fe2+ bound to 4 Ns, other 2 bonds perpendicular to plane of ring available for bonding • In Hb, one of these aHached to N terminus of His, other binds O2.
Structure of heme porphyrin heme (Fe-protoporphyrin IX)
B. Structure and func9on of myoglobin • It is a heme protein present in heart and skeletal muscle • Reservoir for O2 and carrier of O2 in muscle cell • Single polypep.de chain similar to polypep.des in Hb • 1. α-‐helical content: • ~ 80% of pep.de in 8 stretches of α-‐helix Labeled A to H • Terminated by Pro or β-‐bends and loops stabilized by H bonds and ionic bonds.
• 2. Loca9on of polar and nonpolar amino acid residues: • Interior made up of hydrophobic amino acids stabilized by hydrophobic interac.ons • Surface → charged amino acids – form H bonds with water • 3. Binding of heme group: • Heme in crevice lined with non-‐polar amino acids, except 2 His residues • Proximal his9dine – binds directly to Fe2+ of heme • Distal his9dine stabilizes binding of O2 to Fe2+
C. Structure and func9on of hemoglobin • Found exclusively in RBCs → transports O2 • Hb A – predominant form in adults: 4 polypep.de chains -‐-‐ α2β2 • Each subunit – heme-‐binding pocket similar to myoglobin • Can transport O2 and CO2 • O2-‐binding proper.es aﬀected by allosteric eﬀectors, unlike myoglobin
1. Quaternary structure of hemoglobin: • 2 iden.cal dimers: (αβ)1 and (αβ)2 • dimers held together by hydrophobic interac.ons (on contact surfaces of subunits as well as internally) but ionic and H-‐bonding also exist • 2 dimers held together by weak polar bonds • diﬀerent conforma.on in deoxyHb and oxyHb
T and R forms of Hemoglobin T = “taut” → deoxy Hb → low affinity for O2 R = “relaxed” → oxy Hb → high affinity for O2
• a. T form: “taut” form • deoxy form of Hb • 2 αβ dimers joined by ionic and H-‐bonds • low oxygen-‐aﬃnity form of Hb • b. R form: • binding of O2 disrupts some ionic and H-‐ bonds between αβ dimers • “relaxed” form • high oxygen-‐aﬃnity form of Hb
D. Binding of oxygen to myoglobin and hemoglobin • D. Binding of oxygen to myoglobin and hemoglobin • Myoglobin → one heme → binds one O2 • Hb → 4 heme→ binds 4 O2 • Hb binding: degree of satura.on (Y) from 0 to 100% • 1. Oxygen dissocia9on curve: • plot of Y against PO2 • myoglobin : higher aﬃnity for O2 than Hb • P50 is 1 mm Hg for myoglobin and 26 mm Hg for Hb
• a. Myoglobin: • O2 dissocia.on curve hyperbolic • This reﬂects that myo binds single O2 • Mb + O2 MbO2 they exist in equilibrium • Exchange between Hb and Mb, Mb and muscle cells depending on equilibrium • Mb binds O2 released from Hb, releases when O2 drops. Mb then releases the O2 into the muscle cell. This only happens when there is an O2 demand.
• b. Hemoglobin: • O2 dissocia.on curve is sigmoidal • Coopera.ve bind of O2 (increased aﬃnity for Hb with more binding) • Heme-‐heme interac.on: binding of O2 at one heme increases aﬃnity for O2 at others
• E. Allosteric eﬀects • Ability of Hb to bind O2 depends on allosteric (“other site”) eﬀectors: – PO2 – pH of environment – PCO2-‐ an inc will cause the inc in unloading of O2. – 2,3-‐disphosphoglycerate availability • allosteric factors do not aﬀect myoglobin
• 1. Heme-‐heme interac9ons: • structural changes in one heme group transmiHed to others • aﬃnity for last O2 ~300X aﬃnity for ﬁrst O2 • a. Loading and unloading of oxygen: • more O2 can be delivered to .ssues with small changes in PO2 • Graph showing loading and unloading at diﬀerent par.al pressures of O2. Hb alterna.vely carries O2 and CO2 between lungs and .ssues • b. Signiﬁcance of sigmoidal O2-‐dissocia9on curve Compare a hyperbolic curve to a sigmoidal curve • A sigmoidal curve gives increasing aﬃnity of O2 for Hb with increasing par.al pressure while a hyperbolic curve is a straight line in that range.
• 2. Binding of CO2: • Most of the CO2 in the blood is transported as bicarbonate: • CO2 + H2O H2CO3 • H2CO3 HCO3-‐ + H+ • Some CO2 binds to the terminal –NH2 of the α and β chains forming carbaminoHb. • Binding of CO2 stabilizes the “taut” form of Hb (deoxyHb).
• 3. Binding of CO: • CO binds reversibly to the Fe2+ the same way that O2 does • CO + Hb HbCO (carbon monoxy Hb) • Aﬃnity of Hb for CO is 220X aﬃnity for O2 • Binding of CO to Hb increases aﬃnity of remaining sites for O2 • O2 dissocia.on curve shigs to leg (becomes hyperbolic) • > 60% HbCO fatal • treated with O2 therapy
4. Bohr Eﬀect: • Shig of O2 dissocia.on curve to the right with decrease in pH or increase in PCO2 • This translates to a decreased aﬃnity of Hb for O2 under these condi.ons, therefore you unload O2 easier
• a. Source of the protons that lower the pH: • 2 principle sources of protons: – lac.c acid produced by anaerobic metabolism in muscles – increased produc.on of CO2 by cell u.liza.on of O2 through respira.on: • CO2 + H2O H2CO3 H+ + HCO3-‐ – in lungs the equilibrium of this reac.on is towards the leg because CO2 is lost through exhaling • the decreased aﬃnity of Hb for O2 under the Bohr eﬀect condi.ons results is greater oﬀ loading (release) of O2 in the .ssues.
The Effect of CO2 and H+ on O2 Binding Bohr Effect: Increased concentrations of CO2 and H+ promote the release of O2 from hemoglobin in the blood.
How do CO2 and H+ promote the release of O2from hemoglobin?• presence of “salt bridge” • no ionic interaction in in T form R form
CO2 is bound to hemoglobin at protein interfaces, notFe2+ center!
• Summary reac.on for the Bohr eﬀect: • HbO2 + H+ HbH+ + O2 OxyHb DeoxyHb • Equilibrium shigs to the right when H+ conc. increases (decrease in pH), while it shigs to leg when PO2 increases. • The protonated forms of the terminal α-‐ subunit –NH2 groups and His side-‐chains stabilize the T form (deoxy form) of Hb.
• 5. Eﬀect of 2, 3-‐bis-‐ phosphoglycerate(BPG) on oxygen aﬃnity: • Important regulator of Hb binding O2 • Most abundant organic phosphate in RBC (conc. ~ = conc. of Hb) • Synthesized from intermediate of glycolysis • a. Binding of 2,3-‐BPG to deoxyhemoglobin: • Binds to deoxyHb stabilizing it • Decreases aﬃnity of Hb for O2
• b. Binding site of 2,3-‐BPG: • 1 molecule of 2,3-‐BPG binds to a pocket between the β-‐chains in the center of the deoxyHb center • expelled on oxida.on of Hb (pocket disappears) • c. ShiX of oxygen-‐dissocia9on curve: • Blood stripped of 2,3-‐BPG has a high aﬃnity for O2 • 2,3-‐BPG shigs the O2-‐dissocia.on curve to the right allowing decreased aﬃnity of Hb for O2 and eﬀec.ve unloading of O2 in .ssues • similar to Bohr eﬀect but no diﬀerence between lungs and .ssues
• d. Response of 2,3-‐BPG levels to chronic hypoxia or anemia: • 2,3-‐BPG increases in chronic hypoxia • chronic hypoxia can be caused by – pulmonary emphysema or – high al.tudes or – chronic anemia • increased 2,3-‐BPG shigs O2 dissocia.on further to right allowing greater unloading of O2
• e. Role of 2,3-‐BPG in transfused blood: • 2,3-‐BPG essen.al for normal transport func.on of blood • Without normal concs. of 2,3-‐BPG, Hb becomes an O2 trap (doesn’t unload; high aﬃnity) • Blood for transfusion formerly stored in acid-‐citrate-‐ dextrose medium decreased 2,3-‐BPG conc. → “stripped” blood • Body restores conc. of 2,3-‐BPG in 24 – 48 h • 2,3-‐BPG can be restored by adding inosin
Minor HemoglobinsEmbryonic form is Hb Gower 1(ζ2ε2) (yolk sac).HbF - 2 α chains, 2 γ chains (β-chain family) - major form infetus and newborn (fetal liver –2 weeks).HbA - 2 β chains, 2 α chains -major form in adult.Fetal bone marrow beginssynthesizing HbA around 8thmonth.
Steps in globin chain synthesis:1. Transcription2. Modification of mRNA precursor by splicing3. Translation by ribosomes & further modifications (i.e. glycosylation)
Hemoglobinopathies• caused by abnormal structure of Hb• characterized by low levels of normal Hb Sickle-cell anemia (Hemoglobin S disease) Hemoglobin C disease Hemoglobin SC disease Thalassemias – α thalassemia β thalassemia
Sickle-cell anemia (HbS disease)• abnormal β chain. HbS = α2βS2• β chain mutation - glu 6 à val 6• glu is negatively charged, val is nonpolar.• only has effect postnatally because HbF is majorspecies in fetus• symptoms - hemolytic anemia, painful crises,poor circulation, frequent infections• heterozygotes - HbA and HbS both present - 1 in10 African Americans; "sickle cell trait" - nosymptoms, normal life span
Sickle-cell anemia (HbS disease)• glutamic acid is replaced by valine at position 6 of βchain
Symptoms worsen when Hb is in deoxy form - decreased pO2,increased CO2, decreased pH, increased 2,3-BPG
Low solubility of HbScauses aggregation anddistortion of cell shape.
HbS• val instead of glu atposition 6HbA• glu at position 6HbC• lys instead of glu atposition 6HbSC• HbS as well as HbCpresent → 2 bands inelectrophoresis
HbC disease• lys instead of glu at position 6• HbC homozygotes - mild, chronic hemolytic anemia. Not life-threatening HbSC disease • HbS as well as HbC present → 2 bands in electrophresis • usually undiagnosed until infarctive crisis occurs (childbirth, surgery) • can be fatal
Thalassemias• hereditary hemolytic diseases• most common genetic disorder in humans• heterogeneous collection of diseases
β-thalassemias• synthesis of β-chain decreased or absent β-thalassemia minor (or trait) - one normal, one defective β- chain gene. Not life-threatening β-thalassemia major - both genes defective. Normal at birth. Severe anemia by age 1-2. Treatment requires frequent transfusions → Leads to iron overload (hemosiderosis). Death between 15-25 years old. Bone marrow transplant (BMT) is an option.
α-thalassemias• decreased or absent α chain synthesis• severity of disease depends upon the numberof defective α genes:0 defective - normal1 defective - silent carrier of α-thalassemia. Nosymptoms2 defective - α-thalassemia trait - no serioussymptoms3 defective - Hemoglobin H disease - moderatelysevere hemolytic anemiaall 4 defective - hydrops fetalis - fetal death (αchains needed for HbF)
Methemoglobinemia • 1. Forma9on of methemoglobin • Oxida.on of Fe2+ → Fe3+ converts Hb and myoglobin to metHb and metmyoglobin • Cannot bind O2, • Oxida.on by drugs like nitrates, H2O2 or free radicals or muta.on in α-‐ or β-‐chain of globin → methemoglobinopathy (HbM). • a. Reduc9on of methemoglobin: • Normal oxida.on corrected by NADH-‐cytochrome b5-‐ reductase • RBCs of newborns → half the capacity of this enzyme, therefore more suscep.ble to oxida.on
Fibrous proteins are characterized as generally having: • one domina.ng kind of secondary structure (i.e. collagen helix in collagen) • a long narrow rod-‐like structure • low water solubility • a role in determining .ssue/cellular structure and func.on (e.g. collagen, α-kera.n)
Collagen -‐ most abundant protein in body; rigid, insoluble Elas.n -‐ stretchy, rubber-‐like, lungs, walls of large blood vessels, ligaments
Structure of Collagen Tropocollagen is a right-‐handed triple helix formed of α-‐chains.
Structure of Collagen The α-‐chains (individual polypep.des composing tropocollagen) consist of -‐[Gly-‐X-‐Y]-‐ repeats. Proline and hydroxyproline/hydroxylysine are ogen present in the X and Y posi.ons, respec.vely.
Synthesis of collagen • made in ﬁbroblast, osteoblasts (bone), chondroblasts (car.lage) • secreted into ECM • enzyma.cally modiﬁed • aggregate and are cross-‐linked
Biosynthesis of collagen 1. forma.on of pro-‐α-‐chains -‐ contains signal sequence – promotes binding of polysome to RER and secre.on into the cisternae; signal sequence removed 2. some pro and lys residues (in the Y posi.on of gly-‐X-‐Y) are hydroxylated by prolyl hydroxylase and lysyl hydroxylase; needs molecular O2 and reducing agent like ascorbic acid (from vitamin C). 3. glycosyla.on -‐ glucose and galactose added to hydroxylysines; pro-‐α-‐chains join to form procollagen. N-‐ and C-‐terminal extensions form interchain disulﬁde bonds; central triple helix formed because of favorable alignment; Transported to Golgi, packaged, and secreted as procollagen.
Biosynthesis of collagen (cont’d) 4. N-‐procollagen pep.dase and C-‐procollagen pep.dase remove terminal extensions, leaving triple helical collagen (occurs extracellularly). 5. collagen ﬁbrils -‐ form by associa.on of collagen molecules with about a 3/4 overlap with other molecules (staggered, parallel arrays) 5. cross-‐linking -‐ interchain cross-‐links caused by lysyl oxidase (a pyridoxal phosphate and copper-‐requiring enzyme); O2 required; oxida.ve deamina.on of lysines and hydroxylysines; Allysine (aldehyde) reacts with amino group of nearby lysine or hydroxylysine to form interchain cross-‐ link. Very important for tensile strength of collagen.
Ascorbate coenzyme required by prolyl/lysyl hydroxylase in hydroxyla.on step. Vitamin C (ascorbate) deﬁciency results in scurvy (collagen can’t be cross-‐linked).
Cross links formed by lysyl/ Cu2+/ prolyl oxidase vitamin B6 -‐ copper coenzyme Number of cross-‐links increases with age → causes s.ﬀening, decreased elas.city of skin and joints.
Biosynthesis of collagen (con’t) In the ﬁnal step, collagen ﬁbrils form spontaneously from tropocollagen. covalent X-‐links between Allysine and hydroxylysine tropocollagen molecule triple helix of α-‐chains.
Types of Collagen CommonType Representative Tissues disorders Ehlers-Danlos Osteogenesis I Imperfecta skin, bone, tendons, cornea Marfan’s cartilage, intervertebral disks, vitreous II - body blood vessels, lymph nodes, dermis, III Ehlers-Danlos early phases of wound repair Alport’s IV basement membranes Goodpasture’s X - epiphyseal plates
Collagen Degrada.on and Disorders • degrada.on of collagen by collagenase allows remodeling of ECM Ehlers-‐Danlos – hyperextensive joints, hyperelas.city of skin, aor.c aneurisms, rupture of colon, skin hemmorhages due to muta.on in α-‐chains Osteogenesis Imperfecta – briHle bone disease, mul.ple fractures, blue sclera, hearing loss, retarded wound healing
Ehlers-‐Danlos Syndrome Hyperextension of skin
In Utero Radiograph: • crumpled long bones • beaded ribs
Elas.n • rubber-‐like proper.es • connec.ve .ssue protein • lungs, large blood vessels, elas.c ligaments Composi.on: -‐ small nonpolar amino acids (Gly, Ala, Val) -‐ also rich in Pro and Lys -‐ liHle or no OH-‐Pro or OH-‐Lys
Elas.n • 3D network of cross-‐linked polypep.des • cross links involve Lys and alLys • 4 Lys can be cross-‐linked into desmosine • desmosines account for elas.c proper.es
Elas.n Degrada.on and Disorders • in lungs -‐ lung alveolar elas.n in constantly exposed to neutrophil elastase α1-‐AT inhibits elastase thus preven.ng loss of lung elas.city • individuals who are homozygotes for mutant α1-‐AT are very suscep.ble to emphysema
Some nomenclature…Active site = special pocket where substrate bindsSpecificity1. enzymes are specific for a single molecule or a structurally related group of substrates2. usually only 1 enzyme per reaction type
Some more nomenclature…Cofactor = inorganic component needed for enzyme function
Some more nomenclature…Coenzyme = nonprotein small organic componentneeded for enzyme function
Some more nomenclature…Holoenzyme - the enzyme protein plus its cofactorApoenzyme - enzyme protein without its cofactorProsthetic groups – a coenzyme that’s very tightly (usually covalently) attached to the protein, such as heme
How Enzymes Work Enzymes increase the rate of reactions without themselves being altered in the process of substrate conversion to product. This defines a catalyst. Enzymes increase reaction rates by lowering the energy input needed to form a reactant complex that will eventually form product. This occurs via the formation of a complex between enzyme and substrate (ES): k1 k2 E + S ES E + P k-1
Steps in an Enzymatic Reaction1. Enzyme and substrate combine to form a complex.2. Complex goes through a transition state – not quite substrate or product3. A complex of the enzyme and the product is produced.4. Finally, the enzyme and product separate.All of these steps are equilibria.
Effect of pH on enzyme activity Examples of optimum pH
Effect of temperature on enzyme activity • exceeding normal temperature ranges always reduces enzyme reaction rates • optimum temperature is usually 25 - 40 ºC (but not always)
Kinetics• Kinetics is the study of the rate of change ofreactants to products• Velocity (v) refers to the change in conc. ofsubstrate or product per unit time• Rate (k) refers to the change in total quantity (ofreactant or product) per unit time• Initial velocity (v0) is the change in reactant orproduct conc. during the linear phase of a reaction
Michaelis-Menten KineticsThree basic assumptions:1: ES complex is in a steady state, i.e. remains constant during the initial phase of a reaction2: when enzyme is saturated all enzyme is in the form of ES complex3: if all enzyme in ES then rate of product formation is maximal: Vmax = k2[ES]
Michaelis-Menten Kinetics The Michaelis-Menten equation is a quantitative description of the relationship between the rate of an enzyme catalyzed reaction (v1), substrate concentration [S], the M-M rate constant (Km) and maximal velocity (Vmax)
Michaelis-Menten Kinetics Km is equal to the concentration of substrate required to attain half maximal velocity for any given reaction
Lineweaver-Burk Analysis• Lineweaver and Burk manipulated the MMequation by taking its reciprocal values generating adouble reciprocal plot• Leads to a linear graph of the reciprocals ofvelocity and substrate concentration
Enzyme inhibition • many substances can inhibit enzyme activity: substrate analogs toxins drugs metal complexes
Enzyme inhibition - 2 broad classes:Irreversible inhibition• forms covalent or very strong noncovalent bonds• site of attack is amino acid group that participates in the normal enzymatic reactionReversible inhibition• forms weak, noncovalent bonds that readily dissociate from an enzyme• the enzyme is only inactive when the inhibitor is present
Enzyme inhibition Competitive inhibitor • resembles the normal substrate and competes for the same site
Enzyme inhibition Examples of competitive inhibitors: • methanol and ethylene glycol compete with ethanol for the binding sites to alcohol dehydrogenase • methotrexate competes with folic acid for dihydrofolate reductase
Enzyme inhibition Noncompetitive inhibitor • materials that bind at a location other than the normal site • results in a change in how the enzyme performs
Enzyme inhibition Examples of noncompetitive inhibitors: • physostigmine is a cholinesterase inhibitor used in the treatment of glaucoma • captopril is an ACE inhibitor used in treatment of hypertension • allopurinol is a xanthine oxidase inhibitor used to treat gout
Enzyme Inhibition - SummaryCompetitive• Inhibitor binds at substrate site, inhibition is reversible as higher substratecompetes for inhibitor, Vmax unchanged, Km increasedNoncompetitive• Inhibitor binds at site other than substrate, ESI cannot form product, increasedsubstrate does not compete, Km unchanged, Vmax decreased
Enzyme Regulation• Proteolytic cleavage to activate: Enzyme exists in inactive form (zymogen) that isactivated by removal of a short peptide segment ( truncation)• Covalent modification to increase or decrease activity, most common is phosphorylation• Sequestration: enzyme forms inactive polymers• Allosteric (“other site”) regulation, both positive and negative ( homotropic, heterotropic)Induction-upregulation: increase gene expression, synthesis of more enzymemoleculesRepression-downregulation: decrease gene expression, decrease synthesis ofenzyme molecules.
Allosteric enzymesAre regulated by molecules called effectors(modifiers) that bind non-covalently at a siteother than active site. They can alter Vmax orKm or both)1. Homotrophic effectors – when the substrateitself is an effector2. Heterotrophic effector – when the effector isdifferent from a substrate (often it is an end-product - feedback inhibition)
Allosteric enzymes show sigmoid curve(cooperative substrate binding like in Hb)
Enzymes Used in Clinical diagnosesTissue damage: Increased release of tissue enzymes in plasma Enzyme assay is used for both diagnostic and prognostic purpose Eg: ALT – present in the liver will be appearing in the plasma if there is Liver damage or cell necrosis Isoenzymes: Structurally different enzymes but catalyze the same reaction Eg: CK1, CK2, CK3 (creatine kinase, CK MB (CK 2) is present in the heart, its presence in plasma is indicative of myocardial infarction
ALSO: Troponin T & Troponin Iare also released in cardiacdamage. Peaks in 8 – 24hrSensitive and specific for cardiactissue damage