kinetics of enzyme reactions

Kinetics of Enzyme Kinetics of Enzyme ReactionsReactions

Srbová Martina

E + S ES E + Pk1

k-1

kcat

rapid

reversible reaction

slow

irreversible reaction

Rate of the conversion of substrate to products (S P):

v = kcat [ES]

Michaelis-Menten EquationMichaelis-Menten Equation1. 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

kcat =Vmax

[E]t

Vmax [S] v =

[S] + Km

Michaelis constant

Lineweaver-Burk PlotLineweaver-Burk Plot

1 Km 1 1

v Vmax [S] Vmax

= • +

Multisubstrate reactionsMultisubstrate 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 activityEnzyme activityStandard 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

Reversible InhibitionReversible Inhibition

Competitive InhibitionCompetitive Inhibition

E + S ES E+P

EI

+I

Competitive inhibitors bind at substrate binding site and compete with the substrate for the enzyme

no inhibitor

plus inhibitor

Noncompetitive InhibitionNoncompetitive InhibitionE + S ES E+P

EI + S EIS

+I

+I

Noncompetive inhibitors bind at a site other than the substrate binding site

no inhibitor

plus inhibitor

Uncompetitive InhibitionUncompetitive Inhibition

E + S ES E+P

EIS

+I

Uncompetitive inhibitors bind only with the ES form of the enzyme

Irreversible InhibitionIrreversible 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 activityControl of enzyme activity

1. Allosteric enzymes

A B C DE1 E2 E3

Negative feedback /feedback inhibition

2. Covalently modulated enzymes

zymogens Glycogen phosphorylaseundergo cleavage to

produce an active enzyme

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 HexokinaseKm Km liver mostly in the other tissues

not inhibited by Glc- 6P inhibited by Glc- 6P

Thank you for your attentionThank you for your attention