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    Enzymes Enzymes Presentation Transcript

    • DEFINITION OF TERMS Enzymes – special proteins that catalyze biochemical reactions Apoenzyme – protein part of an enzyme; catalytically inactive Cofactors – small, non-protein components  Coenzymes – small organic molecules, often vitamin-derived  Prosthetic group  Metal ions – K+, Mg+2, Zn+2, Mn+2 , Cu+2, Na+, Fe+2 Holoenzyme = Apoenzyme + Cofactors; catalytically active Substrate – the molecule acted upon y the enzyme to form a product Active site of the enzyme – part of the enzyme consisting of a chain of amino acids involved in catalyzing the reactions; generally located in clefts or crevices Allosteric site – additional site where allosteric molecules (stimulators or inhibitors) bind and affect the activity of the active site. Regulatory enzyme – the enzyme that catalyzes the regulatory or committed step of a metabolic pathway
    • DEFINITION OF TERMS Protein APOENZYME part + CofactorNonprotein •Coenzyme part •Prosthetic group •Metal ion HOLOENZYME
    • ENZYME COFACTORS Chemical Vitamin A. Coenzyme Enzyme Groups Precursor Transferred Thiamine Pyruvate dehydrogenase,Pyrophosphate Isocitrate dehydrogenase, α- Aldehydes Thiamine (TPP) ketoglutarate dehydrogenase, (Vit B1) Transketolase, α-ketoacid dehydrogenase Flavin Adenine Succinate dehydrogenase, α- Riboflavin Dinucleotide ketoglutarate dehydrogenase, Electrons (Vit B2) (FAD) Pyruvate dehydrogenase, Nitric oxide synthase Nicotinamide Lactate dehydrogenase; Hydride ion Nicotinic acid Adenine Other dehydrogenases (:H-) (Niacin; B3)Dinucleotide (NAD) Glycogen phosphorylase, Pyridoxine Pyridoxal γ-ALA synthase, Histidine Amino groups (Vit B6)Phosphate (PLP) decardoxylase, Alanine aminotransferase Lipoate Pyruvate dehydrogenase Electrons and Not required in α-Ketoglutarate dehydroge- acyl groups diet nase
    • ENZYME COFACTORS Chemical Vitamin A. Coenzyme Enzyme Groups Precursor Transferred Acetyl CoA Acyl groups Pantothenic Coenzyme A carboxylase acid & other compounds Pyruvate carboxylase, CO2 Acetyl CoA Biocytin carboxylase, Biotin Propionyl CoA carboxylase 5’- Methylmalonyl H atoms and Vit B12deoxycobalamin mutase alkyl groups Thmidylate One-carbon Folic acidTetrahydrofolalate synthase groups
    • ENZYME COFACTORS Cofactor EnzymeB. Inorganic (Metal ionsor iron- sulfur clusters) Zn+2 Carbonic anhydrase, Carboxypeptidase A & B Cu+2 Cytochrome oxidase Mn+2 Arginase, ribonucleotide reductase Mg+2 Hexokinase, pyruvate kinase, glucose 6-phosphatase Ni+2 Urease Mo Nitrate reductase Se Glutathione peroxidase Mn+2 Superoxide dismutase K+ Propionyl CoA carboxylase
    • ENZYME COFACTORS: COENZYMES COOH H -COOH COO TPP Pyruvate S~CoA | Succinate | dehydrogenase | CH2 dehydrogenase H – C | C=O C=O | || | | CH2 C–H CH3 CoA CH3 | | + NAD NADH+ COOH FAD FADH2 H -COOH Pyruvate Acetyl CoA +H Succinate Fumarate1. The oxidation of pyruvate to acetyl CoA via pyruvate dehydrogenase utilizes several coenzymes like thiamine pyrophosphate (TPP), CoA (Coenzyme A) and NAD+ (Nicotinamide adenine dinucleotide); this reaction links the glycolytic pathway with the Krebs Cycle; not a part of the Krebs Cycle proper.2. The oxidation of succinate to fumarate is catalyzed by succinate dehydrogenase with FAD as a coenzyme; FAD accepts 2 H+ from succinate and in the process it is reduced to FADH2 while succinate is converted to fumarate; this reaction is one of the reversible steps of Krebs Cycle.
    • ENZYME COFACTORS:COENZYMES Succinyl CoA Histidine Pyruvate Glycineδ-Aminolevulenate PLP Histidine Biotin- synthase PLP decarboxylase CO2 Pyruvate δ-aminolevulenic acid ATP carboxylase CO2 ADP + several Histamine Pi reactions Oxaloacetate Heme (Fe protoporphyrin IX)1. In the synthesis of heme, succinyl CoA condenses with glycine catalyzed by γ-amino- levulinate synthase with pyridoxal phosphate (PLP) as a coenzyme; this is followed by several enzyme-catalyzed reactions until heme is formed.2. Histidine is decarboxylated into histamine via histidine decarboxylase, with PLP as a coenzyme.3. The carboxylation of pyruvate into oxaloacetate is catalyzed by pyruvate carboxylase, with biotin as a coenzyme; biotin here is a carrier of CO2. Other enzymes that use biotin as a coenzyme include acetyl CoA carboxylase and propionyl CoA carboxylase, hence biotin is a carrier of activated CO2 (as HCO3-) in carboxylation reactions.
    • ENZYME COFACTORS: METAL IONS Glucose Phosphoenolpyruvate (PEP) CO2 ATP ADP Mg+2 Hexokinase/ Mg+2 K+ H 2O Carbonic Pyruvate Glucokinase anhydrase ADP kinase + Pi ATP Zn+2Glucose 6-phosphate Pyruvate H2CO31. The phosphorylation of glucose into glucose 6-phosphate is catalyzed by hexokinase or glucokinase, in the presence of ATP, and Mg+2 as a metal cofactor; this is the 1st step of glycolysis.2. The conversion of phosphoenolpyruvate (PEP) into pyruvate is catalyzed by pyruvate kinase, with K+ as a cofactor metal, aside from Mg+2; this is the last irreversible step of glycolysis.3. The hydrolysis of CO2 into carbonic acid (H2CO3) is catalyzed by carbonic anhydrase, with Zn+2 as a cofactor metal.
    • SIX MAJOR CLASSES OF ENZYMES (IUBMB*, 1964) CLASS EXAMPLE Oxidoreductases Dehydrogenases, Oxidases, Reductases, Peroxidases, Catalases, Oxygenases, Hydroxylases Transferases Transaldolase and Transketolase; acyl, methyl and glucosyl phosphotransferases, Kinases, Phosphomutases, Transaminases Hydrolases Esterases, Glycosidases, Peptidases, Phosphatases, Thiolases, Phospholipases, Amidases, Deaminases, Ribonucleases Lyases Decarboxylases, Aldolases, Hydratases, Dehydratases, Synthases, Lyases Isomerases Epimerases, Isomerases, Mutases, Racemases Ligases Synthetases, Carboxylases*International Union of Biochemistry and Molecular Biology; classification is based by the reactions enzymes catalyze; each class is divided into subclasses.
    • SIX MAJOR CLASSES OF ENZYMES (IUBMB*, 1964) CLASS TYPE OF REACTION CATALYZED Oxidoreductases Transfer of electrons (Hydride ions or H atoms) Transferases Group transfer reactions Hydrolases Hydrolysis reactions (transfer of functional groups to H2O) Lyases Addition of groups to double bonds, or formation of double bonds by removal of groups Isomerases Transfer of groups within molecules to yield isomeric forms Ligases Formation of C-C, C-S, C-O, and C-N bonds by condensation reactions coupled to cleavage of ATP or similar cofactor
    • OXIDOREDUCTASES: DEHYDROGENASES Transfer of electrons and hydrogen atoms from donors (or reductants, hence oxidized) to acceptors (or oxidants, hence reduced). COO- Lactate COO- | dehydrogenase | HO – C – H C=O | | CH3 CH3 L-Lactate NAD+ NADH Pyruvate + H+1. Lactate Dehydrogenase – catalyzes the transfer of 2 H from donor lactate (or reductant, hence oxidized) to acceptor NAD+ (or oxidant, hence reduced); NAD+ is reduced to NADH + H+ (NADH2).2. Oxidation – loss of electrons Reduction – addition of electrons
    • TRANSFERASES: KINASES Transfer of functional groups (like, C-, N- or P-) from O donors to acceptors. O || || C1 - H C1 - H | | H - C2 - OH H - C2 - OH | | OH - C3 - H Hexokinase OH - C3 - H | Glucokinase | H - C4 - OH H - C4 - OH | | H - C5 - OH H - C5 - OH | Mg+2 | H - C6 – OH H - C6 - O – P | | H ATP ADP + Pi H Glucose (donor) (product) Glucose 6- phosphate (acceptor) (product)1. Hexokinase or Glucokinase – catalyzes the transfer of the terminal phosphate group of ATP (donor) to carbon 6 of glucose (acceptor), forming the 2 products: glucose 6- phosphate and ADP.2. This is the 1st step of glycolysis.
    • TRANSFERASES: TRANSAMINASESTransfer functional groups (like C-, N-, or P-) from donors to acceptors; utilize 2 substrates to produce 2 products. substrate COO- C=O COO- COO- | I | | H3N – C – H + (CH2)2 Alanine C=O + H3N – C – O | I transaminase | | CH3 COO- CH3 (CH2)2 L-Alanine α-ketoglutarate Pyruvate |(amino acid) (keto acid) (keto acid) COO- PLP L-Glutamate substrate substrate product (amino acid) product1. Alanine transaminase (an aminotransferase) - catalyzes the transfer of an amino group (NH3) from alanine (an amino acid donor) to α-ketoglutarate (a keto acid acceptor) to form pyruvate (a keto acid) and glutamate (an amino acid) respectively.2. Here, α-ketoglutarate (a keto acid) is converted to glutamate (an amino acid) while alanine (an amino acid) is converted to pyruvate (a keto acid).
    • HYDROLASES: PHOSPHATASES Catalyze cleavage of chemical bonds by addition of H2O, producing 2 products Phosphate bond O O O || || ||-O – P ~ O – P ~ O- + HOH Pyrophosphatase 2 HO – P – O- | | | -O -O O- Pyrophosphate Phosphate (PPi) 2 (Pi)1. Pyrophosphatase - catalyzes the cleavage of a high-energy phosphate bond of pyrophosphate (PPi) in the presence of H2O, forming 2 inorganic phosphates (2 Pi).2. The process is essentially irreversible, with the transfer of –OH from HOH (H2O) to inorganic phosphate (Pi).
    • LYASES: DECARBOXYLASES Add H2O, NH3, or CO2 to and from double bonds NH3+ I + CH2 – CH – COO- CH2 – CH2 – NH3 4 3 DOPA decarboxylaseHO HO OH OH PLP3,4-Dihydroxyphenyl- Dopamine CO2 alanine (DOPA) 1. Dopa decarboxylase catalyzes the removal of –COO- from DOPA to synthesize dopamine and CO2. 2. PLP is a coenzyme of DOPA decarboxylase.
    • ISOMERASES: ISOMERASES & MUTASES Transfer of functional groups or double bonds within the same molecule O Aldehyde H || group | C1 - H H – C1 - OH Keto O Phospho- O | | group || glycerate || H - C2 - OH C2 = O C1 – O- mutase C1 – O- | | | | OH - C3 - H Phosphohexo- OH - C3 - H H – C2 – OH H – C2 – O - P | isomerase | | | H - C4 - OH H - C4 - OH H – C3 – O – P H – C3 – OH | | | | H - C5 - OH H - C5 - OH H H | | H - C6 - O – P H - C6 - O – P 3-phospho- 2-phospho- | | glycerate glycerate H HGlucose 6-Phosphate Fructose 6-Phosphate (Aldose) (Ketose) 1. Phosphohexoseisomerase catalyzes the shift of a double bond from carbon 1 in glucose 6-phosphate to carbon 2 in fructose 6-phosphate (or interconversion of an aldose and ketose sugar). 2. Phosphoglycerate mutase catalyzes the transfer of a phosphate group from carbon 3 in 3-phospholglycerate to carbon 2 in 2-phosphoglycerate.
    • LIGASES: SYNTHETASES & CARBOXYLASESCatalyze the ligation or joining of 2 substrates in the presence of ATP COO- COO- Biotin-CO2 COO- | |H3N – C – H Pyruvate | H3N – C – H CH3 carboxylase CH2 | + NH4+ | (CH2)2 | | Glutamine (CH2)2 | | C=O C=O synthetase C C | | // // COO- ATP ADP COO- O O- O NH2 + Pi Pyruvate Oxaloacetate L-Glutamate L-Glutamine ADP ATP + Pi1. Glutamine synthetase - catalyzes the condensation of glutamate and NH4+ in the presence of ATP to form glutamine, ADP and Pi.2. Pyruvate carboxylase – catalyzes the condensation of pyruvate and CO2 (from biotin) to form oxaloacetate in the presence of ATP; this is the first step in gluconeogenesis – the synthesis of glucose form noncarbohydrate substrates.
    • CHARACTERISTICS OF ENZYMES They are not changed by the reaction they catalyze – although they may be temporarily changed during the reaction; they are neither used up in the reaction nor do they appear as reaction products but are regenerated or recycled They do not change or alter the equilibrium position of the reaction, so they cannot force a reaction that is not energetically favorable (non- spontaneous); equilibrium would be attained rapidly in the presence of an enzyme. They increase reaction rates by decreasing the activation energy- or lowering the energy needed to form a complex of reactants that is competent to produce reaction products; the velocity of the reaction they catalyze is measured by the amount of product formed per unit time. They are highly specific for the reactants or substrates they act on. They are mostly proteins in nature although a small number of RNA- based biological catalysts called ribozymes have been identified.
    • ENZYMES DECREASE THE ACTIVATION ENERGY + + Transition state, S + + ΔG (uncatalyzed) + + ΔG (catalyzed) Energy level Substrates or ΔG + Reactants for the (e.g. CO2 + H2O) reaction Products (H2CO3) Reaction progress1. In a chemical reaction, a substrate (or a reactant) is converted to a product via the formation of an activated or transition state (S≠) which has a higher free energy than does either the substrate or the product. In other words, substrates need a lot of energy to reach a transition state.2. ΔG≠ is the activation energy or Gibbs free energy of activation which represents the difference in free energy between the transition state and substrate.3. In enzyme-catalyzed reactions, the ΔG≠ (or activation energy) is lowered, hence enzymes facilitate the formation of the transition state and consequently reducing the energy needed to form the products→ favorable formation of the products → ↑ reaction rate.
    • MODELS OF ENZYME-SUBSTRATE COMPLEX Lock and Key Model Induced Fit Model 1. Lock and Key Model (Emil Fischer, 1894) - the substrate binds to a site whose shape complements its own, like a key in a lock (or the correct piece in a 3-dimensional jigsaw puzzle); this model is now largely historical because it does not take into account the 3-dimensional flexibility of proteins. 2. Induced Fit Model ( Daniel E. Koshland, Jr., 1958) - the enzyme undergoes a slight conformational change on binding to the substrate, hence enzyme forms a complementary shape or fit after the substrate is bound; more attractive model and replaced the earlier rigid lock and key model.
    • KINETICS OF ENZYME-CATALYZED REACTIONS k1 k2 E+S ES E+P k-1 k-2 Substrate binding Catalytic step1. In typical enzyme-catalyzed reactions, reactant and product concentrations are usually hundreds or thousands of times greater than the enzyme concentration; hence, each enzyme molecule catalyzes the conversion to product of many reactant molecules.2. Enzyme E binds to the substrate S (substrate binding) to form an enzyme substrate complex ES (sometimes called Michaelis complex), with rate constant K1 (K1= the rate constant for the formation of ES).3. The ES complex has 2 possible fates: a. It can dissociate to E and S, with a rate constant of K-1 b. It can proceed to form product P (catalytic step), with a rate constant of K2 (K2 = the rate constant for the conversion of P from the enzyme E); K-2 represents the regeneration of ES from E and P.
    • MICHAELIS-MENTEN EQUATION Vmax [S] Vo = {Km + [S]}Vo = Velocity at any time (moles/time)Vmax = Maximal velocity (or reaction rate)Km = Michaelis constant for the particular enzyme under investigation = (K-1 + K2)/K1[S] = Substrate concentration (molar)1. A quantitative description of kinetics of enzyme-catalyzed reactions.2. Describes how reaction velocity varies with substrate concentration.3. Vo (velocity at any time is = to …etc….4. Vmax = …..5. Km = ….6. [S] = ….7. Velocity of an enzyme-catalyzed reaction is too difficult to measure, hence velocity can be determined by measuring the products formed per unit time.
    • MICHAELIS-MENTEN SATURATION CURVE Vmax │ │ │ │ │ │ Reaction velocity (VO) Vmax C 2 Zero order B A First order Km │ │ │ │ │ │ │ │ │ Substrate concentration [S]1. A graphical representation of Michaelis-Menten Equation, it is a basic model for non-allosteric enzymes, describing how reaction velocity varies with substrate conc.2. Reaction velocity (Vo; no. of moles of product formed/ unit time) varies with substrate concentration, hence Vo linearly increases as substrate conc. increases and begins to level off and approaches a maximum velocity (Vmax) at higher substrate concentrations.3. A hyperbolic curve (rectangular hyperbola) is formed since maximum is reached asymptotically.4. At Point A where substrate conc. is less than km or {[S] < Km}, reaction velocity appears to be proportional to the substrate concentration→ only a portion of the enzyme molecules are bound to the substrate and the reaction is said to be first order.
    • MICHAELIS-MENTEN SATURATION CURVE Vmax │ │ │ │ │ │ Reaction velocity (VO) Vmax C 2 Zero order B A First order Km │ │ │ │ │ │ │ │ │ Substrate concentration [S]5. At Point B, exactly half of the enzyme molecules are in an ES (enzyme-substrate) complex at any instant and the reaction velocity rate is exactly 1/2 of Vmax {[S]=Vmax/2}; thus the Michaelis-Menten constant (Km) is substrate concentration [S] yielding a velocity of Vmax/2 (or substrate concentration at half-maximal velocity).6. At high substrate concentration (near Point C) when the [S] is > Km, Vo = Vmax, i.e., reaction velocity rate is maximal; reaction is said to be in zero order because further increases in substrate concentration will not result to an increase in velocity since almost all of the enzymes are bound and saturated with substrates.
    • SIGNIFICANCE OF KM1. It is the substrate concentration at which half of the active sites of the enzyme are filled up.2. It is an inverse measure of the affinity of the substrate for the enzyme: a. The lower the km, the higher is the affinity → enzyme requires only a small amount of substrates to become saturated, hence the lower is the substrate concentration needed to achieve a given rate → Vmax is reached at relatively low substrate concentration.. b. The higher the km, the lower is the affinity → enzyme requires an increased amount of substrate to become saturated, hence the higher is the substrate concentration needed to achieve a given rate → Vmax can be reached only at high substrate concentration.
    • LINEWEAVER-BURKE DOUBLE RECIPROCAL PLOT 1 V Intercept Slope = Km Vmax on X-axis = -1 Km 1 Intercept on Y-axis = Vmax 1/S 1. In the Michaelis-Menten Curve, line is hyperbolic → difficult to estimate Vmax since it is an asymptote and value is never reached with any finite substrate concentration → also difficult 1 to determine Km of the enzyme. 2. Lineweaver-Burke Double Reciprocal Plot linearizes the Michaelis-Menten Equation → more precise way to measure Vmax and Km; it is easier to draw the best straight line through a set of points than to estimate the best fit of points to a curve. 3. The reciprocal of reaction velocity, 1/V, is plotted on the y axis. 4. The reciprocal of substrate concentration, 1/[S], is plotted on the x axis. 5. The slope of the line is Km/Vmax; Y intercept is 1/Vmax; X intercept is -1/Km. 6. To attain Vmax, multiply [S] by 100.
    • INHIBITION OF ENZYMATIC REACTIONSReversible a. Competitive b. Non-competitive c. UncompetitiveIrreversible
    • REVERSIBLE INHIBITION1. Competitive Inhibition a. Inhibitor strongly resembles the substrate → binds to the enzyme’s active site →substrate is prevented from binding with the enzyme. b. Relieved by increasing the substrate concentration.2. Noncompetitive Inhibition a. Inhibitor binds to enzyme other than at the active site →structural change in the enzyme’s active site → substrate cannot bind to the enzyme no products formed. b. Inhibition cannot be reversed by increasing substrate concentration since inhibitor cannot be driven from the enzyme.
    • REVERSIBLE INHIBITONInhibitor Type Binding Site on Enzyme Kinetic Effect Competitive Inhibitor binds specifically at active or Vmax unchanged; Inhibitor catalytic site, where it competes with Km increased to substrate for binding; inhibition is reversed reach a given by increasing substrate concentration. velocity.Noncompetitive Inhibitor binds E or ES other than at the Vmax decreased Inhibitor active or catalytic site; substrate binding proportionately unaltered but ESI complex cannot form due to inhibitor to structural change in the enzyme →↓ concentration; catalytic power no products formed; Km unchanged inhibition cannot be reversed by increasing (since substrate substrate concentration since inhibitor can still bind to cannot be driven from the enzyme. the enzyme). Uncompetitive Inhibitor binds only to ES complexes at Inhibitor locations other than catalytic site; Vmax decreased substrate binding modifies enzyme Km decreased structure, making inhibitor-binding site available; inhibition cannot be reversed by increasing substrate concentration; rare in occurence.
    • LINEWEAVER-BURKE DOUBLE RECIROCAL PLOT IN THE PRESENCE OF AN INHIBITOR Noncompetitive inhibitor CompetitiveType of Vmax Km inhibitorInhibition Uncompetitive inhibitorCompetitive Same ↑ 1 [V]Noncompetitive ↓ Same Uninhibited enzymeUncompetitive ↓ ↓ 1 Vmax -1 1 Km [S]
    • KINETICS FOR AN ALLOSTERIC ENZYME1. Allosteric enzymes – consist of multiple subunits and multiple active sites that bind small regulatory molecules (either positive or negative effectors) at allosteric sites different from the catalytic active site  conformational change to the active site.2. Do not obey Michaelis-Menten kinetics  sigmoid curve (instead of hyperboilc curve).3. This curve indicates that the binding of a substrate to one active site can affect the properties of the other sites in the same enzyme molecule.4. Binding then becomes cooperative, i.., the binding of a substrate to the active site of the enzyme facilitates substrate binding to the other sites, hence a sigmoidal plot of the Vo vs. [S].
    • REGULATION OF ENZYME ACTIVITY Feedback Inhibition Allosteric (Non-covalent) Modification Covalent Modification Zymogen Activation Induction or Repression of Enzyme Synthesis
    • FEEDBACK INHIBITION Original Precursor(s) Enzyme 11. Substrates or precursors are converted to final 1 products via a series of enzyme-catalyzed Enzyme 2 reactions. 22. These products then inhibit an earlier enzyme Enzyme 3 and thus shuts down the 3 whole series, preventing the accumulation of Enzyme 4 intermediates in the pathway.3. Hence, products inhibit Enzyme 5 their own synthesis. Final Products
    • ALLOSTERIC MODIFICATIONAllosteric modulator (activator or inhibitor) Binds to regulatory or allosteric site Conformational change in the regulatory enzyme Effect is transmitted to the active site Change in shape of the active site Altered enzyme activity (↑or↓)
    • Protein COVALENT MODIFICATION kinase Pyruvate ATP ADP dehdrogenase ATP kinase ADP Enzyme Pyruvate Pyruvate I dehydrogenase dehydrogenase P Enzyme OPO3- I (active) (inactive) OH HPO4= H2O Phosphoprotein Pyruvate phosphatase Pi dehdrogenaseH O phophatase 21. Involves either addition or removal of phosphate groups from the enzyme via protein kinases or phosphoprotein phosphatase, with ATP as phosphate donor; some enzymes are activated via phosphorylation; others are inactivated by dephosphorylation.2. Pyruvate dehydrogenase catalyzes the conversion of pyruvate to acetyl CoA; a kinase catalyzes the phosphorylation of the enzyme, making it phosphorylated and inactive.3. On the other hand, pyruvate dehydrogenase phosphatase catalyzes the dephosphorylation of the enzyme, making it more active; hence this enzyme is inactivated by dephosphorylation and activated by phosphorylation.4. In general, enzymes that catalyze biosynthetic reactions are active when dephosphorylated and inactive when phosphorylated (ex. Glycogen synthase is active when dephosphorylated; inactive when phosphorylated); those that catalyze degradative reactions are active when phosphorylated and inactive when dephosphorylated.
    • ZYMOGEN ACTIVATION:BLOOD COAGULATION Intrinsic Pathway Extrinsic Pathway XII XIIa VIIa VII XI XIa Tissue Factor (III) IX IXa X Xa X1. Blood coagulation represents a series of sequential interactive events that lead to the repair of the vascular system following V Va XIII injury via the formation of a fibrin clot. Prothrombin (II) Thrombin2. Upon activation, the individual clotting (IIa) XIIIa factor serves as an enzyme to convert the inactive zymogen to the succeeding Soluble active form “a”. Fibrinogen (I) Fibrin3. For example: FXa converts the zymogen Prothrombin to its active form Thrombin, which in turn converts the inactive zymogen Insoluble Fibrinogen to its active form Fibrin (hence a Fibrin cascade).
    • INDUCTION OR REPRESSION OF ENZYME SYNTHESIS ↑ Blood glucose ↓ Blood glucose levels levels (Starvation) (Well-fed state) ↑ Insulin ↑ Glucagon ↑ Synthesis of ↑ Synthesis of key enzymes involved key enzymes involvedin glucose degradation in glucose synthesis
    • FACTORS AFFECTING ENZYME ACTIVITY Temperature pH Substrate concentration Co-factors
    • EFFECT OF TEMPERATURE │ │ │ │ │ │ │ │ │ │ │ Optimum T Reaction velocity (Vo) Increasing enzyme activity Heat inactivation of the enzyme │ │ │ │ │ │ │ │ │ │ │ │ │ │ │ │ 10 20 30 40 50 60 70 80 Temperature (oC)1. Velocity of an enzyme-catalyzed reaction increases with increase in temperature until the optimum is reached; optimum temperature is the temperature at which the rate of the reaction is maximal; for most human enzymes, optimum temperature is between 40-60o C.2. The increase in velocity before the optimum temperature is due to increased kinetic energy of the substrate and the enzyme → more collision between enzyme and substrate → more binding.3. Beyond optimum temperature or with further increase in temperature → ↓ velocity due to inactivation secondary to denaturation of the enzyme, specifically destruction of the tertiary structure or unfolding of the enzyme; plotting temperature vs. velocity, a curve slightly skewed to the right is obtained.
    • EFFECT OF pH Optimum pH1. Before and beyond optimum pH, there is a decreasing velocity of an enzyme- catalyzed reaction due to enzyme denaturation (because the structure of the catalytically active site of the protein molecule depends on the ionic character of the amino acid side chains).2. Optimum pH refers to the pH at which the velocity of the reaction is highest.3. Plotting pH vs. velocity, a bell-shaped curve will be obtained.
    • EFFECT OF CO-FACTORS:Chlorides, Bromides, IodidesCofactors increase the rate of enzyme-catalyzed reactions
    • EFFECT OF SUBSTRATE CONCENTRATION Vmax Reaction velocity (V) Substrate concentration [S]1. For a given conc. of an enzyme, the velocity of a reaction ↑es as the substrate conc. increases until maximal velocity (Vmax) is reached.2. Thereafter, further increases in the substrate conc. will no longer increase the velocity because all the enzymes have been saturated by the substrate.3. Sometimes, however, the addition of excessive amounts of substrates after Vmax will ↓ reaction vel. due to the fact that there are so many substrates competing for the active sites on the enzyme surfaces that they block the sites prevent any other substrate molecules from occupying them drop in velocity since all of the enzymes present is not being used.
    • CARDIAC ENZYMES AS MARKERS FOR ACUTE MYOCARDIAL INFARCTION Aspartate aminotransferase1. Troponin (Troponin T and Troponin I isoforms) - regulatory proteins involved in myocardial contractility very specific and preferred markers for detecting myocardial cell injury, as in MI  therefore not present in the serum of healthy individuals. Rises 3-6 hours after injury; peaks in 12-16 hrs; stays elevated in 5-14 days.2. Creatine Kinase a. Begins to rise 4-6 hours after MI; peak at 24 hrs; returns to normal in 3-5 days. b. Isoenzymes: i. CK-MM fraction = found in skeletal muscle ii. CK-MB fraction = found in heart muscle iii. CK-BB = found in the brain c. May be increased in other conditions: physical exertion, postoperatively, convulsions, delirium tremens, etc; hence not diagnostic for MI unless the CK-MB fraction is being assayed: rises in 3- 4 hours after MI; peak 12-14 hrs later and returns to normal in 2 days.
    • CARDIAC ENZYMES AS MARKERS FOR ACUTE MYOCARDIAL INFARCTION Aspartate aminotransferase3. Lactate Dehydrogenase a. Peak level about 36- 40 hrs after MI and thus of diagnostic value in patients admitted > 48 hrs after infarction. c. Levels return to normal in 5-14 days b. No longer used to diagnose MI  found also in other tissues like liver, RBCs, skeletal muscles, and a variety of organs. 4. Aspartate Aminotransferase, AST a. Rise within 8 hrs after MI; peak at 24-36 hrs; returns to normal level within 3-7 days. b. Not diagnostic for MI since the enzyme is also found in hepatocytes.