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BCH210H1 (352)
Lecture

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Department
Biochemistry
Course
BCH210H1
Professor
Roman Melnyk
Semester
Fall

Description
BCH210 – Enzymes and Hemoglobin Lecture 1 – Enzyme Regulation Enzymes and Hemoglobin: - Introduction to enzymes - Active sites and enzyme regulation - Enzyme reactions and kinetics - Enzyme inhibition and mechanisms - Hemoglobin Introduction to enzymes Essential questions - What are enzymes, and what do they do? - What characteristic features define enzymes? - Can the rate of an enzyme-catalyzed reaction be defined in a mathematical way? - What equations define the kinetics of enzyme-catalyzed reactions? - What can be learned from the inhibition of enzyme activity? - What is the kinetic behavior of enzymes catalyzing bimolecular reactions? - How can enzymes be so specific? - Are all enzymes proteins? - Is it possible to design an enzyme to catalyze any desired reaction? Virtually all reactions in cells are mediated by enzymes - Enzymes catalyze thermodynamically favorable reactions, causing them to proceed at extraordinarily rapid rates - Living systems use enzymes to accelerate and control the rates of vitally important biochemical reactions - Enzymes are the agents of metabolic function Enzyme properties 1. They are highly specific for their reactants or substrates (S) 2. Many exhibit stereospecificity 3. Specificity of the reaction 4. Enzyme (E) means “present in cells” (does not generate by-products) 5. Sumner crystallized urease in 1926 6. Almost all enzymes are proteins 7. Classification, nomenclature, kinetics, regulation of E activity coenzymes (required by E for full activity) Enzyme classification ENZYME CLASSIFICATION ENZYME CLASSIFICATION 1. OXIDOREDUCTASES: Oxidation-Reduction Reactions Lactate Dehydrogenase 1. Oxidoreductases: oxidation-reduction reactions (lactate dehydrogenase) Coenzyme Reduced Coenzyme - *Enzy*Enzymes called oxidases, dehydrogenases, reductases - NAD+ iNAD+ is nicotinamide adenine dinucleotide - *One substrate is oxidized, one is reduced - Oxidation: loss of electrons *One substrate is oxidized, one is reduced - ReducOxidation: loss of electronsansfer Reactions Reduction: gain of electronAlanine Transaminase 2. Transferase: group transfer reactions (alanine transaminase) - *Group transfer reactions, kinases carry out phosphorylation reactions - Eg. Carnitine acyltransferaseKinases carry out phosphorylation rxns e.g. Carnitine acyltransferase Fatty acyl CoA: fatty acid in an activated form, attached to CoA (CoenzymeA) Enzyme transfers acyl group to the molecule l-carnitine, producing acylcarnitine and CoA - Fatty acyl CoA: fatty acid in an activated form, attached to CoA (coenzyme A) - Enzyme transfers acyl group to the molecule 1-carnitine, producing acylcarnitine and CoA - Kinase3. HYDROLASES: Catalyze Hydrolysis, H 0 also a2Substrate Pyrophosphatase 3. Hydrolases: catalyze hydrolysis, H O2also a substrate (pyrophosphatase) - Largest group - Hydrolytic reactionstions - Eg. Pyrophosphatase uses water to break PPi (pyrophosphate)phate) - ThisThis reaction ENZYME CLASSIFICATIONydrolysis of anhydride bond links phosphates) - Prot(that links phosphates)es, esterases are also hydrolases - Examples are trypsin, chymotrypsin *Proteases, lipases, nucleases, esterases are also hydrolases 4. Lyases: creates a double bond or addition to a double bond (pyruvare decarboxylase) - *Lyases*Lyases can also be called synthasescatalyze addition re(CO 2ns) - Eg. Fatty acid synthase makes fatty acids with a release of CO 2to provide energy) ENZYME CLASSIFICATIONon reactions) 5. Isomerases: catalyze an isomeration (bisphosphogylcerate) - Simplest reaction acid synthase makes fatty acids with a release2of CO 5. ISOMERASES:(to provide energy) Catalyze an Isomerization Bisphosphoglycerate Mutase ENZYME CLASSIFICATION 6. LIGASES: Catalyze the Joining of Two Molecules 6. Ligases: catalyze the joining of two molecules (glutamine synthetase) Source of Chemical Energy - *Ligation of joining of two molecules, requires energy - Input (eg. Uses ATP, a molecule that carries energy) energy Input (e.g. usesATP, a molecule that carries energy) - Fatty acyl CoA synthetase (a ligase)gase): - ATP drives formation of link between CoA and fatty acid - ATP ▯ AMP + PPi + energy (ATP: carrier of chemical energy in its phosphate bonds)TP ▯ AMP + PPi + energy (ATP: carrier of chemical energy in its phosphate bonds) What characteristic features define enzymes? - Catalytic power is defined as the ratio of the enzyme-catalyzed rate of a reaction to the uncatalyzed rate - Specificity is the term used to define the selectivity of enzymes for their substrates - Regulation of enzyme activity ensures that the rate of metabolic reactions is appropriate to cellular requirements - Enzyme nomenclature provides a systematic way of naming metabolic reactions - Coenzymes and cofactors are nonprotein components essential to enzyme activity - Enzymes can accelerate reactions as much as 10 over uncatalyzed rates - Urease is a good example: 4 o Catalyzed rate: 3 x 10 /-10 o Uncatalyzed rate: 3:10 /sec o Ratio is 1/10 (very high catalytic power) Enzyme specificity - Enzymes selectivity recognize proper substrates over other molecules - Enzymes produce products in very high yields – often much greater than 95% (∴ no wasteful by-products) - Specificity is controlled by structure – the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield 1.Urease1. Urease + 2 3 + 2. Alcohol dehydrogenase 2.Alcohol Dehydrogenase: + + 2. Alcohol Dehydrogenase: ENZYME SPECIFICITY + + 3. Hexokinase 3. Hexokinase: ENZYME SPECIFICITY 4. Proteases 4. Proteases: OR ENZYME SPECIFICITY 3 4 - + 3 4 5. TrypsinEster 5. Trypsin Active site (makes up less than 1% of total enzyme but enzymes are big… why?) 1. There are specialized region in the enzyme necessarily for S binding and catalysis 2. Typically active site 3D cleft/crevasse within enzyme formed by distant regions polypeptide region brought together 3. Active site have unique microenvironment excludes water – except hydrolases 4. Active sites tend to have 2-3 key residues needed for catalysis ACTIVE SITE Active site N C Trp63 Asp52 Glu35 Asp101 Lysozyme Trp108 Trisaccharide - N-acetyl glucosamine = GlcNAc - N-acetyl muramic acid = MurNAc N-Acetyl Glucosamine = GlcNAc N-Acetyl MuramicAcid = MurNAc ACTIVE SITE ACTIVE SITE R 1 R2 Binding Site 3 For S Induced Fit Lock and Key (E) (S) Lecture 2 ENZYME REGULATION Enzyme regulation 1. Substrate Availability: 1. Substrate availability + Injury Enzyme Activation Phospholipid in Membrane ArachidonicAcid Degraded Phospholipid Cyclooxygenase, 2 (COX) Eicosanoids + + + Thrombus Formation VasconstrictionPain, Fever ENZYME REGULATION 2. Covalent Modification: 2. Covalent modification - Reversible - Phosphorylation - P ▯ ATP - Ser, Tyr, Thr (-OH) Phosphorylase Kinase (Inactive)ase b (Active)ylase P 3. Allosteric control - Fastest way to regulate an enzyme ENZYME REGULATION + Glucose-1-Phosphate - “other” = alloION Glycogen Energy 3. Allost-ric Different from active site 3.  Allosteric  Control…continued: Active Site Feedback Inhibition E S E S E Modulator Thr 1 Ile - Allosteric Site Energy Status Control Active Site Glucose Active Site S PFK-1 c S PFK-1 Modulator r r c PFK-1 ADP Allosteric Site Activated r=Regulatory Subunit + + + c=Catalytic Subunit Energy Requiring Reactions ATP▯ADP ENZYME REGULATION- Clinical Insight Energy▯ ATP Enzyme regulation – Clinical insight (“Gout”) Loss of Allosteric Control Phosphoribosylpyrophosphate synthetase (PRS) PRS Ribose-5-Phosphate + ATP ------> 5-phosphoribosyl-pyrophosphate + AMP - Purine Nucleotides (A/G) Degradation ENZYME REGULATION 4. Zymogens, Proenzymes: URATE 4. Zymogens, proenzymes Duodenum Hexapeptide + ( NH -Val-(Asp) -Lys-COO ) 3 4 Enzyme reactions - Specific catalyst that speed up speed of equilibrium by decreasing activation barrier A B E A B Enzyme kinetics Can the rate of an enzyme-catalyzed reaction be defined in a mathematical way? - Kinetics is the branch of science concerned with the rates of chemical reactions - Kinesis ▯ “movement” or rate - Enzyme kinetics seeks to determine the maximum reaction velocity that enzymes can attain and binding affinities for substrates and inhibitors - Analysis of enzyme rates yield insights into enzyme mechanisms and metabolic pathways - This information can be exploited to control and manipulate the course of metabolic events ENZYME REACTIONS (Vo) Enzyme reactions (V )TIO0S (Vo) E A B ColorleRed E Linearity from ENZYME REACTIONLinear Chemical kinetics provides a foundation for exploring enzyme kinetics - Consider a reaction of overall stoichiometry as shown: 𝑆 → 𝑃 𝑑 𝑃 −𝑑 𝑠 𝑣 = 𝑑𝑡 = 𝑑𝑡 −[𝑠] 𝑣 = = 𝑘[𝑠] 𝑑𝑡 - The rate is proportional to the concentration of S ENZYME KINETICS Enzyme kinetics .05 0.1 0.2M The Michaelis-Menten equation is the fundamental equation of enzyme kinetics - Louis Michaelis and Maud Menten’s theory - It assumes the formation of an enzyme-substrate complex - It assumes that the ES complex is in rapid equilibrium with free enzyme - Breakdown of ES to form products is assumed to be slower than: 1. Formation of ES and 2. Breakdown of ES to re-form E and S MICHAELIS-MENTEN KINETICS Michaelis-Menten Kinetics k 1 -1,2k rate constants k2= kcat 1. Rate of P production: v=k [E2] 1) R2. Rate of ES formation: k [E][S] 2) Rate of ES formation: k [1][S]1 3) Rate of ES breakdown: (k + k )[ES]ES2 Steady state: k1[E][S]=(k -1 )2ES] 2 ▯▯▯▯▯▯ Steady State: k1[E][S] ▯ (k-1 k 2 [ES] ▯▯ ▯ [▯] Michaelis-Menten equation: 𝑣 = ▯ ▯▯▯   Michaelis Constant : ▯▯▯[▯] Derive the Michaelis-Menten Equation Michaelis-Menten Equation: DERIVE THE MICHAELIS-MENTEN EQUATION Pages: 411-413 ENZYME KINETICS Enzyme Kinetics If [S] - KIf [S]=Km, V= ½Vmax PHYSIOLOGICALCONSEQUENCE OF Km Understanding Km - The “kinetic activator constant” - K ms a constant - K ms a constant derived from rate constants - K ms, under true Michaelis-Menten conditions, an estmate of the dissociation constant of E from S - Small Kmmeans tight binding; highmK means weak binding Physiological consequence of m Alcohol Ethanol Dehydrogenase + + Acetaldehyde - + + Acetaldehyde Acetate Understanding V mas - The theoretical maximal velocity - V maxis a constant - V maxis the theoretical maximal rate of the reaction – but it is never achieved in reality - To reach V max would require that all enzyme molecules are tightly bound with substrate - V maxis asymptotically approached as substrate is increased RECIPROCALOF MICHAELIS- MENTEN EQUATION Reciprocal of Michaelis-Menten equation y = mx + b The turnover number defines the activity of one enzyme molecule - A measure of catalytic activity - K cathe turnover number, is the number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate - If the M-M model fits, k 2k cat max/Et - Values of k catange from less than 1/sec to many millions per sec TURNOVER NUMBER: kcat Turnover number: k cat Lecture 3 – Enzyme inhibition Reversible inhibition REVERSIBLE INHIBITION - Competitive: (I binds to E only) CLASSICAL COMPETITIVE INHIBITION (I binds to E orNON-CLASSICALCOMPETITIVE INHIBITION ClassCompetitive: (I binds to E only) Non-classical competitive inhibition Non-competitive: (I binds to E or ES) COMPETITIVE INHIBITION COMPETITIVE INHIBITION Competitive inhibition k2 COMPETITIVE INHIBITION Succinof Competitive Inhibitionssic Example Non-competitive inhibition NON-COMPETITIVE INHIBITION NON-COMPETITIVE INHIBITION k 2 NON-COMPETITIVE INHIBITION IRREVERSIBLE ENZYME INHIBITION IRREVERSIBLE ENZYME INHIBITION Irreversible enzyme inhibition ASA - COX, O2 COX Serine2CH O+ Endoperoxide Eicosanoids ASA, AcetylsalicylicAcid + + + Thrombus FormatVasconstrictiPain, Fever Initial Wounding Response (Injury) Serine-CH O- C-CH COX 2 = 3 O COX Phospholipase IRREVERSIBLE ENZYME INHIBITIONCox (Inactive) Phospholipid in Membrane ALLOSTERIC REGULATION Allosteric regulation Aspartate Transcarbamoylase (ATCase) ATCase ALLOSTERIC REGULATION Regulation of Pyrimidine Nucleotide Synthesis in E. coli ALLOSTERIC REGULATION ALLOSTERIC REGULATION ALLOSTERIC REGULATION ALLOSTERIC REGULATION Structure  of  AT3ase2 …6 6 2  c + 3 r ▯ c r ALLOSTERIC REGULATION ALLOSTERIC REGULATION ATP ALLOSTERIC REGULATION ATCase c Subunit Alone Hyperbolic MECHANISMS OF ENZYMES Lecture 4 – Enzyme mechanisms and cofactors Mechanisms of enzymes Chemical Modes of Enzymatic Catalysis MECHANISMS OF ENZYMES MECHANISMS OF ENZYMES MECHANISMS OF ENZYMES D C E F MECHANISMS OF ENZYMES MECHANISMS OF ENZYMES MECHANISMS OF ENZYMES MECHANISMS OF ENZYMES Ribonuclease A (RNase A) #1 21 20 -S-S- = Cystine Disulfide Bridge #1-20AA = S Peptide Subtilisin #21-124AA = S Protein #124 1 o 21 3 C 20 RNase A + Subtilisin RNase S (Active) 124 1 S Peptide S Protein 20 (Inactive) (Inactive) 21 MECHANISMS OF ENZYMES MECHANISMS OF ENZYMES 124 (12 or 119) 5’ RNA RNaseA 3’ -CH2-COO- Pyrimidine Sites for RNase A Binding: On  3’  Side  of  C  and  U (12 or 119)5’---CGG U AAG CG U AGA CG CAGG UAC GU A---3’ CHEMICAL MODIFICATION OF RNase A Sites of RNase AAttack: MECHANISMS OF ENZYMES MECHANISMECHANISM OF RNA CLEAVAGE BY RNaseMECHANISMS OF ENZYMES MECHANISMS OF ENZYMES MECHANISMS OF ENZYMES MECHANISMS OF ENZYMES Serine Proteases MECHANISMS OF ENZYMES CATALYTIC TRIAD OF CHYMOTRYPSIN Serine Protease Mechanism - A mixture of covalent and general acid-base catalysis 102 - Asp functions only to orient His 57 57 MECHANISM OF CHYMOTRYPSIN- - His acts as a general acid CATALYZED CLEAVAGE and base - Ser 195forms a covalent Catalytic triad of chymotrypsin. The imidazole ring of His-57 removes the proton from the hydroxymethyl side chain of Ser-195 (to which it is bond with peptide to be hydrogen-bonded), thereby making Ser-195 a powerful nucleophile. This cleaved other hydrogen-bonded partner, the buried ▯-carboxylate group of Asp- its 102. - Covalent bond formation MEturns a trigonal C into a tetrahedral C 193 195 - The tetrahedral oxyanion intermediate is stabilized by N-Hs of Gly and Ser MECHANISMS OF ENZYMES E + S E - S E - S E – T1 E – T1 MECHANISMS OF ENZYMES E – T1 Amine Produ1t (P ) MECHANISMS OF ENZYMES Acyl-E + 1 Acyl-E + 2 O Acyl-E + 2 O MECHANISMS OF ENZYMES E – 2 E – T2 E – P 2 MECHANISMS OF ENZYMES Transition-State Stabilization in the Serine Proteases Carboxylate Product (P2) • The chymotrypsin mechanism involves two tetrahedral oxyanion intermediates. Transition-state stabilization in the serine proteases - The chymotrypsin mechanism involves two tetrahedral oxyanion intermediatese stabilized by a pair of amide groups that is - These intermediates are stabilized by a pair of amide groups that is termetermed  the  “oxyanion   hole.” MECHANISMS OF ENZYMES Substrate Specificities: Binding Pockets COFACTORS/COENZYMES Cofactors/coenzymes (Mg , Ca , Fe , Co )YMES (Organic) LDH Coenzyme Reduced COFACTORS/COENZYMES Coenzyme COFACTORS/COENZYMES COFACTORS/COENZYMES E- E- Succinate Dehydrogenase (SDH) COFACTORS/COENZYMES COFACTORS/COENZYMES Adenine High Energy Anhydride Links Ribose Adenosine Triphosphate (ATP) (Flavin Adenine Dinucleotide) Enzyme based diseases and syndromesES AND SYNDROMES DISEASE ENZYME SUBSTRATE CLINICAL INVOLVED SYMPTOMS Hemolytic Anemia G-6-P Dehydrogenase G-6-P Decreased RBC stability Immunodeficiency Adenosine Adenosine Combined T/B cell Deaminase immunodeficiency Albinism Tyrosinase Tyrosine Pigment absent in skin, hair, eyes Fructosuria Fructokinase Fructose Benign, autosomal recessive Fructose-1,6- Fructose-1, 6- Fructose-1, 6- Hypoglycemia, bisphosphate bisphosphatase bisphosphate acidosis Deficiency Lactose IntolerancLactase Lactose Bloating, nausea, abdominal cramps Tarui’s  disease PhosphofructokinaseFructose-6- Cramps on phosphate exercise, hemolysis Lecture 5 – Hemoglobin - A classic example of allostery - Hemoglobin and myoglobin are oxygen transport and storage proteins - Compare the oxygen binding curves for hemoglobin and myoglobin - Myoglobin is monomeric; hemoglobin is tetrameric - Mb: 153 AA, 17,200 MW - Hb: 2 α chains of 141 residues, 2 β chains of 146 residues HEME PROSTHETIC GROUP Heme prosthetic group 2+ Fe is coordinated by His - Iron interacts with six ligands in Hb and Mb - Four of these are the N atoms of the porphyrin - A fifth ligand is donated by the imidazole side chain of amino acid residue His - When Mb or Hb bind oxygen, the O mole2ule adds to the heme iron as the sixth ligand - The O m2lecule is tilted relative to a perpendicular to the heme plMYOGLOBIN Myoglobin - Mb is a monomeric heme protein - Mb polypeptide 2+radles” the heme group - Fe in Mb is Fe - ferrous iron – the form that binds oxygen - Oxidation of Fe yields 3+ charge – ferric iron - Mb with Fe is called metmyoglobin and does not bind oxygen Red blood cells Hemoglobin O 2INDING TO HEME O 2INDING TO HEME O 2inding to heme O BINDING CURVES 2 O 2inding curves O 2INDING CURVES Cooperative binding of oxygen influences hemoglobin function - Mb, an oxygen storage protein, has a greater affinity for oxygen at all oxygen pressures - Hb is different – it must bind oxygen in lungs and release it in capillaries - Hb becomes saturated with O i2 the lungs, where the partial pressure of 2 is about 100 torr - In capillaries, p2 is about 40 torr, and oxygen is released from Hb - The binding of O 2o Hb is cooperative – binding of oxygen to the first subunit makes binding to the other subunits more favorable The physiological significance of the HB:O inte2action - Hb must be able to bind oxygen in the lungs - Hb must be able to release oxygen in capillaries - If Hb behaved like Mb, very little oxygen would be released in capillaries - The sigmoid, cooperative oxygen binding curve of Hb makes it a physiological COOPERATIVE BINDIactions possible INFLUENCES HEMOGLOBIN FUNCTION Cooperative binding of oxygen influences hemoglobin function Fe movement by less than 0.04 nm induces the conformation - In deoxy-Hb, the iron atom2+ies out of the heme plane by about 0.06 nm - Upon O bi2ding, the Fe atom moves about 0.039nm closer to the plane of the heme - As if the O 2s drawing the heme iron into the plane - This may seem like a trivial change, but it’s biological consequences are far-reaching 2+ - As Fe moves, it drags His and the helix with it - This change is transmitted to the subunit interfaces, where conformation changes lead to the rupture of salt bridges 2, 3-BPG BINDING TO HB HAS IMPORTANT PHYSIOLOGICAL SIGNIFICANCE 2,3-BPG binding to Hb has important physiological significance The structure, in ionic form of BPG or 2,3- bisphosphoglycerate, an important allosteric effector of Hb 2,3-bisphosphoglycerate - An Allosteric effector of hemoglobin - In the absence of 2,3-BPG, oxygen binding to Hb follows a rectangular hyperbola - The sigmoid binding curve is only observed in the presence of 2,3-BPG - Since 2,3-BPG binds at a site distant from the Fe where oxygen binds, it is called an allosteric effector 2,3-BPG binding to Hb has important physiological significance 2,3-BISPHOSPHOGLYCERATE - The “inside” story… - Where does 2,3-BPG bind? o “Inside” o In the central cavity - What is special about 2,3-BPG? o Negative charges interact with 2 Lys, 4 His, 2 N-termini - Fetal Hb – lower affinity for 2,3-BPG, higher affinity for oxygen, so it can get oxygen from mother + H promotes dissociation of oxygen from hemoglobin + - Binding of O t2 Hb is affected by several agents, including H , CO , and 2hloride ions + - The effect of H is particularly importan+ - Deoxy-Hb has a higher affinity for H than oxy-Hb - Thus, as pH decreases, dissociation of O fro2 hemoglobin is enhanced + - Ignoring th+ stoichio+etry of O an2 H , we can write HbO +2H ↔ HbH + O 2 HB O S2TURATION CURVES AT DIFFERENT pHs Hb O s2turation curves at different pHs 23 + The antagonism of O bind2ng b+ H is termed the Bohr effect - The effect of H on O bin2ing was discovered by Christian Bohr (the father of Neils Bohr, the atomic physicist) - Binding of protons diminishes oxygen binding - Binding of oxygen diminishes proton binding - Important physiological significance CO a2so promotes the dissociation of O from Hb 2 - In addition, some CO is 2irectly transported by hemoglobin in the form of carbamate - (-NHCOO ) - Free α-amino groups of Hb react with CO reversi2ly: • Catalyze chemical rxns - + R-NH + 3• They
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