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 + B P + Q

Nomenclature: by Clelandsubstrates A, B, C, D,.....etcproducts P, Q, R, S,......etcinhibitors I, J, K,......etcenzyme complex E, F, G(stable complex) enzyme complex EA(unstable transitory complex)

enzyme complex EAB EPQ(central complex)

E : free enzymeF : covalent attachementenzyme complex

1) Sequential Reaction• Ordered sequential

• Random sequential

Order sequential mechanism:

A B P Q

E EA (EAB EAP) EQ E

(b). Theorell-Chance mechanism:steady state concentration of central complexs are low.

A B P Q

E EA EQ E

*It may be impossible for B to bind until after A binds and promotes aconformational change in the enzyme that exposes the B binding site.

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.

Métodos de estudio

• Estudios en velocidad inicial

• Inhibición por productos

• Inhibición por inhibidores de fondo de saco

• Estudios de intercambio isotópico

Estudios en velocidad inicial

• Se varian ambos sustratos en concentraciones no saturantes

Kinetics of Enzyme-catalyzed Reactions Involving Two 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/A

[B]

= V1AB

KiaKb + KaB + KbA + AB

1/

1/B

[A]

Kia= cte de disociacion para A

Ka y Kb son las ctes de Michaelis

para A y B

Kinetics of Enzyme-catalyzed Reactions Involving Two or more Vary Substrates

A + B P + Q

2. Parallel Pattern: An irreversible step intervenes between the timesof combination of the two substrates in the mechanism.

1/

1/A

[B]1/

1/B

[A]

= VAB

KaB + KbA + AB

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 está saturante (50 veces Km).

Product Inhibition in Multi-Substrate SystemsThe Random Sequential Mechanism

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:

Substrates bind in no specified order;

Products release in no specified order.

Not allowed: EA + P <=> (EAP)EB + Q <=> (EBQ)

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

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

E.sucrose E.glucose.fructose E-glucose E.glucose-1-P