Kinetics of Enzyme Reactions Srbová Martina E+S k1 k-1 ES kcat rapid E+P slow reversible reaction irreversible reaction Rate of the conversion of substrate to products (S → P): v = kcat [ES] Michaelis-Menten Equation The ES complex is in a steady state All of the enzyme is converted to the ES complex Rate of formation of the products will be the maximum rate possible Vmax = kcat[E]total Turnover number number of molecules of substrate that one molecule of the enzyme can convert to product per unit time V max kcat = [E]t v= Vmax [S] [S] + Km Michaelis constant Lineweaver-Burk Plot v = Km Vmax • [S] + Vmax Multisubstrate reactions Ternary-complex mechanism Random mechanism Two substrates A and B can bind in any order P,Q - products Ordered mechanism Binding of A is required before B can bound Ping-pong mechanism Substrate A reacts with E to produce product P which is released before the second substrate B will bind to modified enzyme E´ The substrate B is then converted to product Q and the enzyme is regenerated Enzyme activity Standard unit of enzyme activity (U) [ µmol / ] - amount of enzyme that convert µmol substrate per 1min SI unit Katal (kat) [mol /s] - amount of enzyme that convert mol substrate per 1s Factors which effect enzyme activity temperature optimum for human enzymes is between 35 – 45 °C pH Reversible Inhibition Competitive Inhibition E+S ES E+P + I plus inhibitor EI no inhibitor Competitive inhibitors bind at substrate binding site and compete with the substrate for the enzyme Noncompetitive Inhibition E+S plus inhibitor no inhibitor ES + + I I EI + S E+P EIS Noncompetive inhibitors bind at a site other than the substrate binding site Uncompetitive Inhibition E+S ES E+P + I EIS Uncompetitive inhibitors bind only with the ES form of the enzyme Irreversible Inhibition   Irreversible inhibitors cause covalent modification of the enzyme Toxins: e.g Amanitin (Amanita phaloides)  Diisopropylfluorophosfate (DFP) - binds to the serine in the active site ⇒ deactivation of ezyme eg inhibition of acetylcholine esterase  Penicillin inhibits bacterial transpeptidase Control of enzyme activity Allosteric enzymes Negative feedback /feedback inhibition A E1 B E2 C E3 D Covalently modulated enzymes zymogens undergo cleavage to produce an active enzyme Glycogen phosphorylase Isozymes - catalyze the same reaction - differ in AA sequences, catalytic acitivity (substrates/coenzymes affinity ) Lactate dehydrogenase tetrameric, types of subunits M, H M4, M3H, M2H2, MH3, H4 Glucokinase x ↑Km liver not inhibited by Glc- 6P Hexokinase ↓Km mostly in the other tissues inhibited by Glc- 6P Thank you for your attention [...]... with the ES form of the enzyme Irreversible Inhibition   Irreversible inhibitors cause covalent modification of the enzyme Toxins: e.g Amanitin (Amanita phaloides)  Diisopropylfluorophosfate (DFP) - binds to the serine in the active site ⇒ deactivation of ezyme eg inhibition of acetylcholine esterase  Penicillin inhibits bacterial transpeptidase Control of enzyme activity 1 Allosteric enzymes Negative... Negative feedback /feedback inhibition A E1 B E2 C E3 D 2 Covalently modulated enzymes zymogens undergo cleavage to produce an active enzyme Glycogen phosphorylase 3 Isozymes - catalyze the same reaction - differ in AA sequences, catalytic acitivity (substrates/coenzymes affinity ) Lactate dehydrogenase tetrameric, 2 types of subunits M, H M4, M3H, M2H2, MH3, H4 Glucokinase x ↑Km liver not inhibited