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Kinetics of Enzyme Reactions

Kinetics of Enzyme Reactions. Srbová Martina. k 1. k cat. rapid reversible reaction. slow irreversible reaction. k -1. E + S ES E + P. Rate of the conversion of substrate to products (S  P): v = k cat [ ES ]. V max [ E ] t. k cat =. V max [ S ]

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Kinetics of Enzyme Reactions

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  1. Kinetics of Enzyme Reactions Srbová Martina

  2. k1 kcat rapid reversible reaction slow irreversible reaction k-1 E + S ES E + P Rate of the conversion of substrate to products (S  P): v = kcat[ES]

  3. Vmax [E]t kcat = Vmax [S] v = [S] + Km Michaelis constant Michaelis-Menten Equation 1. The ES complex is in a steady state. 2. All of the enzyme is converted to the ES complex. 3. 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

  4. = • + Lineweaver-Burk Plot 1 Km 1 1 v Vmax[S] Vmax

  5. Multisubstrate reactions 1. 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

  6. 2. 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.

  7. Enzyme activity Standard unit of enzyme activity (U) [mol / min ] • amount of enzyme that convert 1 mol substrate per 1min SI unit Katal (kat) [mol /s] - amount of enzyme that convert 1 mol substrate per 1s Factors which effect enzyme activity • temperature • optimum for human enzymes • is between 35 – 45 °C pH

  8. Reversible Inhibition

  9. E + S ES E+P + I plus inhibitor no inhibitor EI Competitive Inhibition Competitive inhibitors bind at substrate binding site and compete with the substrate for the enzyme

  10. E + S ES E+P + I + I plus inhibitor no inhibitor EI + S EIS Noncompetitive Inhibition Noncompetive inhibitors bind at a site other than the substrate binding site

  11. E + S ES E+P + I EIS Uncompetitive Inhibition Uncompetitive inhibitors bind only with the ES form of the enzyme

  12. 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

  13. A B C D E2 E3 E1 Control of enzyme activity • Allosteric enzymes Negative feedback /feedback inhibition

  14. 2. Covalently modulated enzymes zymogens Glycogen phosphorylase undergo cleavage to produce an active enzyme

  15. 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 Hexokinase Km Km liver mostly in the other tissues not inhibited by Glc- 6P inhibited by Glc- 6P

  16. Thank you for your attention

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