cleland cinetica
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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
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