Enzyme of ICBB - Enzymes • • • • • • • most...

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Unformatted text preview: Enzymes • • • • • • • most enzymes are proteins, except RNA accelerates chemical reaction enzyme NOT used up in the process high specificity highly regulated, e.g by phosphorylation activity depends on pH, temperature, etc. holoenzyme = aproprotein (protein) + cofactor (non-protein) 1 Enzymes • enzymes affect reaction rates, NOT equilibrium • enzymes stabilize intermediates by lowering their free energy • enzyme activities are regulatable – allosteric or covalent modification 2 Basic equation of catalysis k1 k2 • E+S k-1 ES E+P E = enzyme S = substrate ES = enzyme . substrate complex P = product • Km (Michaelis-Menten constant) Km = k-1 +k2 +k k1 • smaller Km => higher affinity for substrate 3 Why is Km a measure of enzyme’s affinity k+1 E+S k-1 ES → + k+1 k+2 k+2 E+P Km = k-1 If Km = k-1 k+1 k+2 << k+1 = [E] [S] [ES] = Dissociation constant of ES Therefore, Km is a measure of the “strength of ES complex ↑ Km => ES unstable => weak binding => ↓affinity 4 Active site • active site of an enzyme is the region that binds the substrate (and the cofactors, if any) – specificity of an enzyme depends on the active site • active site is small relative to the whole enzyme • active site is a 3-dimensional entity – not formed by linear amino acid sequence – e.g. lysozyme’s active site: a.a. 35, 62,63,101 & 108 • active site binds to substrate via weak interactions – charge, H-bonds, van der Waal’s forces, hydrophobic Hinteractions 5 Active site models • “Lock-and-key” model – Emil Fisher, 1890 – active site’s shape is a perfect match of substrate • Induced fit model – Daniel E. Koshland, 1958 – active site’s shape is complementary ONLY after binding substrate; i.e. active site’s shape changes upon binding to substrate 6 Lock and key model Substrate and active site have exact same shape i.e. complementary to each other 7 Induced fit model 1. 2. 3. Substrate and active site NOT complementary to each other. Enzyme changes shape upon binding substrate. Active site only complementary to that of the substrate only after substrate is bound 8 Equilibrium ratio (Keq) • ratio of product : substrate at equilibrium Keq = [P] / [S] • determined by the free energy difference between product and substrate (ΔG) R=gas const. ΔG = -RT ln Keq T=temp. ↑ ΔG (more negative) => ↑ [P] /[S] • enzymes do not change Keq , enzymes only affect how fast equilibrium is reached 9 Reaction rate (V) Reaction • how fast substrate is disappearing V = k[S] ; Vmax = V at highest [S] • k determined by activation energy ΔG‡ k = κ T e -ΔG‡ /RT h κ = Boltzmann const. h = Planck’s const. R = gas const. T = temp (in K) • enzymes increase reaction rate by lowering activation energy ↓ ΔG ‡ => ↑ k => faster reaction rate => 10 Binding energy & catalytic power of enzymes • E + S interaction => energy is released (binding energy) => stabilizes the intermediate complex => ↓ activation energy (ΔG ‡) activation ↓ ΔG‡ => ↑ k => faster reaction rate => => • binding energy is derived from weak, non-covalent interactions between E and S • E active sites are complementary to transition state, not to S 11 12 13 Experimental studies of enzyme kinetics • Change [S] => measures initial velocity Vo • Vo vs [S] Vo = Vmax [ S ] K m + [S ] (Michaelis & Menten equation) • M & M eqn can be derived to other eqns e.g. Lineweaver-Burke (1/V0 vs 1/[S]) Lineweaver- 14 Michaelis-Menten plot: Vo = Vmax [ S ] K m + [S ] 15 Michaelis-Menten equation – Et = E + ES – at any time, there are two forms of enzyme: E (free) and ES (bound with substrate) – at low [S], most enzymes are at the free E form. Therefore, the rate of reaction will be proportional to [S] – at high [S], most enzymes are at the bound ES form, i.e. enzymes are “saturated” with substrate and further increase in [S] will have no effect on reaction rate. Vmax is observed at that [S]. 16 Lineweaver-Burk plot Michaelis-Menten plot Vo = Vmax[S] Km +[S] 17 Lineweaver-Burk plot • • • • Based on Michaselis-Menten plot Double reciprocal plot (1/Vo vs. 1/[S]) A linear plot Better than Michaelis-Menten plot – Easier to determine Km and Vmax and 18 Inhibition • Competitive • Non-competitive • Uncompetitive 19 Competitive inhibition • Inhibitor (I) competes with substrate (S) for active site • Higher Km (lower affinity for substrate) • Constant Vmax (high enough [S], outcompetes I for binding to active site) 20 21 Non-competitive inhibition • • • • • “shape” of I ≠ “shape” of S binding of I does not block binding of S enzyme inactivated when I is bound usually constant Km apparent Vmax is lowered (Vmax = kcat[Et]) Java applet to simulate different kinds of inhibition:22 http://www.hort.purdue.edu/rhodcv/hort640c/ho05000.htm 23 Uncompetitive inhibition • I only binds to ES complex • Changes in both Km and Vmax – Ration of Km to Vmax remains unchanged 24 25 Enzymes in diagnosis • Amylase; acute pancreatitis • Alkaline phosphatase; liver disease, biliary obstruction • Aspartate transaminase; liver disease, myocardial infarction • Alanine transaminase; liver disease • Creatine kinase; myocardial infarction • γ-glutamyl transferase (γGT); biliary obstruction • α1-antitrypsin; emphysema 26 Regulation of enzyme activity • Sequential pathway of metabolism –A→B→ C→D E1 E2 E3 Feedback inhibition – At least one enzyme is regulatable • regulatory enzyme • Usually the first enzyme (E1); most efficient way to control (E1); the whole pathway – Other enzymes in the sequence: • Large excess of catalytic activity (E2 , E3) (E2 E3) – Last product inhibits the first enzyme • Feedback inhibition 27 Regulatory enzymes • Activity changes according to signals – Including external signal and product inhibition • Regulatory enzymes usually have 2 subunits – Catalytic subunit • Carries enzyme activity – Regulatory subunit • Does not have enzyme activity • Regulate active subunit’s enzyme activity according to environmental signals 28 Regulatory enzymes • Regulation can be in several ways – Allosteric regulation of enzyme • Conformation changed upon non-covalent binding to modulator non• Fast response – Reversible covalent modification of enzyme • By modifying amino acids – e.g. phosphorylation: adding a phosphate group to serine – Slow response – Activation by proteolytic cleavage of precursor enzyme • Proenzyme => enzyme – e.g. in blood clotting factors , digestive enzymes 29 Allosteric enzymes M C R reversible S C Active R C R Inactive – Regulatory subunit (R) • Allosteric enzyme contains two subunits • Carries modulator ( ) binding site • Binding of modulator induces conformational changes in catalytic subunit e.g. Inactive => active • Substrate binding site can now accept substrate ( ) – Catalytic subunit (C) • Carries substrate binding site • Respond to changes rapidly by binding /releasing modulator 30 Covalent modification • Modifying groups are added covalently to the enzyme – Phosphate, AMP (adenosine monophosphate), UMP, ADP, methyl groups – Mediated by modifying enzymes • Most famous example: phosphorylation – Phosphorylase b <=> phosphorylase a (inactive) (active) – Active enzyme hydrolyzes glycogen to glucose 31 Zymogen • Activation by proteolytic cleavage of the enzyme’s precursor (zymogen) • Irreversible • e.g.enzymes in digestive system – Chymotrypsin (from chymotrypsinogen) chymotrypsinogen) • Cleaved by trypsin – Trypsin (from trypsinogen) trypsinogen) • Cleaved by enteropeptidase • Cleavage enzyme usually inhibited – By binding to an inhibitor – e.g. pancreatic trypsin inhibitor inhibits trypsin 32 Glycogenolysis • Glycogen: storage form of glucose in animals • Glycogen store in body – Liver (> 10% of liver weight = glycogen) • Main storage of glycogen in the body • Purpose: for exporting glucose to blood for other tissues’ use – Muscle • Purpose: glucose only for muscle’s own use • Glycogenolysis = degradation of stored glycogen 33 Glycogen structure http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.2912 34 LIVER Glycogen Glycogen phosphorylase Glucose 1-phosphate Phospho glucomutase Phosphate group Glucose Glucose 6-phosphate Glucose-6phosphatase Glucose Blood glucose EXPORTED TO OTHER TISSUES Glycolysis 35 MUSCLE Glycogen Glycogen phosphorylase Glucose 1-phosphate Phospho glucomutase Glucose 6-phosphate Glycolysis 36 Regulation of Glycogen phoshorylase • Phosphorylation of glycogen phosphorylase – – GPb <=> GPa (phosphorylated, more active) Catalyzed by 1. phosphorylase kinase 1. • ↓ blood glucose → ↑ Glucagon from pancreas →↑ cAMP (cyclic →↑ AMP) in liver → ↑ Protein kinase A (PKA) activity → phosphorylate phosphorylase kinase (↑ activity) → phosphorylate glycogen phosphorylase →↑ GPa form →↑ Add phosphate group Stimulated by Ca2+ (induced by glucagon) ↑ Glucagon→ ↑ cAMP (cyclic AMP) → ↑ Protein kinase A → phosphorylation of PPI (Phosphoprotein phosphatase inhibitor) → ↑ PPI activity → ↑ inhibition of phosphoprotein phosphatase → ↓ PP-1 activity → ↑ GPa form PP- • • • 2. phosphoprotein phosphatase (PP-1) (PP- • • ATP, Glucose, Glucose-6-phosphate Glucose– – – – Inhibits GPb or GPa Becomes “Tense” form (less active) Stimulates GPb or GPa Becomes “Relaxed” from (more active) 37 AMP ...
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