Kinetics of Enzyme Reactions Srbov Martina ES k

Kinetics of Enzyme Reactions Srbová Martina

E+S k 1 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 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 of molecules of substrate that one molecule of the enzyme can convert to product per unit time Vmax kcat = [E]t v= Vmax [S] + Km Michaelis constant

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

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

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.

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

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 l Toxins: e. g. Amanitin (Amanita phaloides) l l Diisopropylfluorophosfate (DFP) - binds to the serine in the active site deactivation of ezyme eg. inhibition of acetylcholine esterase l Penicillin inhibits bacterial transpeptidase

Control of enzyme activity 1. Allosteric enzymes Negative feedback /feedback inhibition A E 1 B E 2 C E 3 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 M 4, M 3 H, M 2 H 2, MH 3, H 4 Glucokinase x Km liver not inhibited by Glc- 6 P Hexokinase Km mostly in the other tissues inhibited by Glc- 6 P

Thank you for your attention