1. Kinetics of Enzyme Reactions Srbová Martina

2. E + S ES E + P k1k1 k -1 k cat rapid reversible reaction slow irreversible reaction Rate of the conversion of substrate to products (S  P): v = k cat [ES]

3. 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. V max = k cat [E] total Turnover number number of molecules of substrate that one molecule of the enzyme can convert to product per unit time k cat = V max [E] t V max [S] v = [S] + K m Michaelis constant

4. Lineweaver-Burk Plot 1 K m 1 1 v V max [S] V max = +

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

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

11. Uncompetitive Inhibition E + S ES E+P EIS +I+I 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. Control of enzyme activity 1. Allosteric enzymes A B C D E1E1 E2E2 E3E3 Negative feedback /feedback inhibition

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

15. 3. Isozymes - catalyze the same reaction - differ in AA sequences, catalytic acitivity (substrates/coenzymes affinity..) Lactate dehydrogenase tetrameric, 2 types of subunits M, H M 4, M 3 H, M 2 H 2, MH 3, H 4 Glucokinase x Hexokinase  K m  K m livermostly in the other tissues not inhibited by Glc- 6Pinhibited by Glc- 6P

16. Thank you for your attention