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M1 – Biochemistry Enzymes I – III Dr. Sato-Bigbee 1 Enzymes can increase the rate of a chemical 2 reaction by a factor of 1011 or higher E+S Enzymes increase the speed at which reactions reach equilibrium but they do not change the equilibrium constant ES P+E Ea: Energy of activation Enzymes speed up reactions by decreasing Ea 3 Why do we need enzymes??? 4 Why do we need enzymes??? Speed Speed: Specificity Enzymes make the speed of reactions compatible with life Control 5 6 Why do we need enzymes??? Speed Specificity: B D (toxic product or molecule without function) A C E (beneficial molecule) 7 8 Why do we need enzymes??? Speed Specificity Control: Inhibited by D End product A E1 B E2 C E3 D 9 Enzyme unit: amount of activity that catalyzes the transformation of 1 umol of substrate / minute 10 active site - binds the substrate -has the amino acid groups that participate in the reaction Specific activity: number of enzyme units/mg of protein (umoles substrate /min /mg protein) Reaction velocity: amount of substrate modified per minute (umoles substrate / min) 11 12 Glucokinase specificity 13 14 15 16 Cofactors Molecules used by enzymes to increase the number of functional groups that participate in the reaction 1) Metal ions 2) Organic coenzymes Thiamine pyrophosphate Important in decarboxylation reactions 17 18 Michaelis-Menten kinetics E + S ↔ [ES] → E + P 19 Km: reflects the affinity of an enzyme for a particular 20 substrate The higher the Km , the lower the affinity for the substrate A E1 B E2 C E3 D E4 E Km for E2 >>> Km for E1, E3, or E4 E2 is the rate limiting enzyme For a particular enzyme and substrate, Km is a constant 21 23 a limiting rate enzyme in a pathway has a high Km 22 24 -1/Km competitive inhibitor likehaving havingless less substrate) (is(is like substrate) Enzyme inhibitors 25 Vmax remains the same Km (apparent Km) increases noncompetitive inhibitor havingless lessenzyme) enzyme) (is (is likelike having Vmax is decreased 26 Km remains the same Irreversible inhibitors Combine with or destroy a functional group that is essential for enzyme activity Several are pharmacologically very important Uncompetitive inhibition (like having less S and less E) 27 28 acetylcholine acetylcholinesterase choline + acetate (Aricept) Penicillin is an irreversible inhibitor of the 29 bacterial enzyme glycopeptide transpeptidase 30 Allopurinol: suicide inhibitor of xanthine oxidase Inhibition of xanthine oxidase by allopurinol 31 for the treatment of gout 32 - Genetic control: regulates the “amount” of enzyme - Induction: activation of the gene encoding an enzyme - Repression: inhibition of the gene encoding an enzyme 33 35 Regulation of glycogen phosphorylase by phosphorylation 34 36 Zymogens or proenzymes: inactive enzyme precursors Cleavage of fibrinogen and clot formation 37 Triggered by interaction of plasma proteins with damaged blood vessel Triggered by tissue factor Exposed on damaged tissue 38 Regulatory Enzymes Allosteric Enzymes: Do not follow Michaelis-Menten kinetics (hyperbolic), they show sigmoideal plots Have two or more subunits The substrate binding sites exhibit co-operativity Are modified by reversible non-covalent binding of regulators Allosteric activators lock the enzyme in a conformation that has high affinity for the substrate Blood coagulation cascade: sequential activation of proenzymes 39 Allosteric inhibitors shift the enzyme to a conformation 40 that has less affinity for the substrate Regulatory Enzymes Allosteric Enzymes: Do not follow Michaelis-Menten kinetics (hyperbolic), they show sigmoideal plots Have two or more subunits The substrate binding sites exhibit co-operativity Are modified by reversible non-covalent binding of regulators Allosteric activators lock the enzyme in a conformation that has high affinity for the substrate 41 Allosteric inhibitors shift the enzyme to a conformation 42 that has less affinity for the substrate 43 44 Regulatory Enzymes Allosteric Enzymes: Do not follow Michaelis-Menten kinetics (hyperbolic), they show sigmoideal plots Have two or more subunits The substrate binding sites exhibit co-operativity Are modified by reversible non-covalent binding of regulators Allosteric activators lock the enzyme in a conformation that has high affinity for the substrate Aspartate transcarbamylase 45 Allosteric inhibitors shift the enzyme to a conformation 46 that has less affinity for the substrate Isoenzymes they catalyze the same reaction they have different amino acid sequence and properties feedback inhibition their characteristics may be related to their function in specific tissues clinically important diagnostic tool 47 48 Isoforms of lactate dehydrogenase Isoenzymes of creatine kinase are used to diagnose myocardial infarction LDH 1 (H4) : heart, kidney, red cells LDH 2 (H3 M): heart, kidney, red cells LDH3 (H2 M2): brain, pancreas creatine kinase: LDH 4 (H M3): lung, spleen CK-1 (BB): brain CK-2 (MB): heart CK-3 (MM): skeletal muscle and heart 49 LDH 5 (M4): skeletal muscle, liver 50