Kinetic vs Chemical Mechanism

An enzyme kinetic mechanism is the order of

substrate addition and product release in an
enzyme catalyzed reaction
A chemical mechanism is the chemical
pathway of conversion of S P, including the
structures of any intermediates

Bi-substrate Reactions
The Michaelis Menten model of enzyme kinetics
was derived for single substrate reactions
The majority of enzymatic reactions have multiple
substrates and products
Bi-substrate reactions account for ~ 60% of the
known enzymatic reactions.

Cinetica CLELAND
Biochim. Biophys. Acta (1963) 67,104-137

67, 173-187

67, 188-196

Substrate Addition / Product Release

The order of substrate addition and product release in
most enzymatic reactions follow two reaction
mechanism
Sequential reaction - all substrates must bind to the
enzyme before the reaction occurs and products are
released
Ordered sequential
Random sequential
Theorell- Chance
Ping-pong reaction - one or more products are
released before all substrates have been added and an
alternate stable enzyme form, F, is produced in the
half reaction

A +
Nomenclature:

by Cleland

substrates A, B, C, D,.....etc
products
P, Q, R, S,......etc
inhibitors
I, J, K,......etc
E : free enzyme
enzyme complex E, F, G
F : covalent attachement
(stable complex)
enzyme complex
enzyme complex
EA
(unstable
transitory complex)
enzyme complex
(central complex)

EAB

EPQ

1) Sequential Reaction
Ordered sequential

Random sequential

Order sequential mechanism:

A

E
EA
(EAB
EAP)
EQ
E
*It may be impossible for B to bind until after A binds and pro
conformational change in the enzyme that exposes the B bind

(b). Theorell-Chance mechanism:steady state concentration

central complexs are lo
A

EA

EQ

example: liver alcohol dehydrogenase.

An Alternative way of Portraying the

Ordered, Single-Displacement
Reaction

This is another view of ordered sequential.

2) Ping-pong Reaction

An Alternative Presentation of the

Double-Displacement (Ping-Pong)
Reaction

Other views of the ping-pong mechanism.

Mtodos de estudio
Estudios en velocidad inicial
Inhibicin por productos
Inhibicin por inhibidores de fondo de
saco
Estudios de intercambio isotpico

Estudios en velocidad inicial

Se varian ambos sustratos en
concentraciones no saturantes

Kinetics of Enzyme-catalyzed Reactions Involving T

or more Vary Substrates

A
+
B
P
+
Q
1. Intersecting Pattern:
indicates sequential combination of both
substrates prior to release of a product.
1/
1/
[B]
[A]

1/A

V1AB
KiaKb + KaB + KbA + AB

1/B

Kia= cte de disociacion para A

Ka y Kb son las ctes de Michaelis
para A y B

Kinetics of Enzyme-catalyzed Reactions Involving T

or more Vary Substrates

A
+
B
P
+
Q
2. Parallel Pattern:
An irreversible step intervenes between the
of combination of the two substrates in the
mechanism.
1/
1/
[A]
[B]

1/A

VAB
KaB + KbA + AB

1/B

WNK1 kinase
Peptido +ATP -> Peptido-P + ADP

UDP-glucose + galactose-1-P ->

Glucose-1-P + UDP-galactose

Methods Enzymol. 1979;63:467-86.

Requisitos
Tener un inhibidor competitivo para cada
sustrato
Estos inhibidores deben ser de fondo de
saco (dead-end), al unirse a la enzima no
se forman productos

Mecanismo Bi-Bi al azar

Inhibidor competitivo para A

Mecanismo Bi-Bi al azar

Inhibidor competitivo para B

Mecanismo Bi-Bi ordenado

Inhibidor competitivo para A

Mecanismo Bi-Bi ordenado

Inhibidor competitivo para B

INHIBICION POR PRODUCTOS

Irreversibilidad
Una etapa en la cual se agrega un
sustrato a la enzima es irreversible si el
sustrato est saturante (50 veces Km).

Product Inhibition in Multi-Substrate Systems

The Random Sequential Mechanism

Substrates bind in no specified order;

Products release in no specified order.

In a typical reaction of this type

the two substrates are bound to the
active site and a section of one
substrate is transferred to the other
to create the products:

Not allowed:

EA + P <=> (EAP)
EB + Q <=> (EBQ)

EB + P <=> (EPB) should form readily:

EA + Q <=> (EAQ) may or may not form

depending on sterics of the transferred side group:
NO

YES, but weaker?

Isotope exchange studies-1

Rate of exchange between a radiolabeled substrate and a product

under equilibrium conditions
First simple test: if exchange occurs between a substrate and a
product when enzyme (+) but second substrate (-) ping-pong
mechanism...
e.g. Sucrose phosphorylase
Isotope exchange btw sucrose (S1) and fructose (P1) (no S2 and P2)

Sucrose

fructose

Pi

G-1-P

E
E.sucrose

E.glucose.fructose

E-glucose

E.glucose-1-P