Kinetics of Enzyme Reactions

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
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 V
max
kcat =
[E]t

v=

Vmax [S]
[S] + Km
Michaelis
constant


Lineweaver-Burk Plot
1
v

=

Km
Vmax

•

1
[S]

+

1
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) [ µmol / min ]
-

amount of enzyme that convert 1 µmol 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 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
1.

Allosteric enzymes

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 by Glc- 6P

Hexokinase
↓Km
mostly in the other tissues
inhibited by Glc- 6P


Thank you for your attention