两步酶促反应稳态速率的pH依赖性

PH-dependence of the steady-state rate of a two-step enzymic reaction.

作者信息

Brocklehurst K, Dixon H B

出版信息

Biochem J. 1976 Apr 1;155(1):61-70. doi: 10.1042/bj1550061.

DOI:10.1042/bj1550061

PMID:7241

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1172802/

Abstract

The pH-dependence is considered of a reaction between E and S that proceeds through an intermediate ES under "Briggs-Haldane' conditions, i.e. there is a steady state in ES and [S]o greater than [E]T, where [S]o is the initial concentration of S and [E]T is the total concentration of all forms of E. Reactants and intermediates are assumed to interconvert in three protonic states (E equilibrium ES; EH equilibrium EHS; EH2 equilibrium EH2S), but only EHS provides products by an irreversible reaction whose rate constant is kcat. Protonations are assumed to be so fast that they are all at equilibrium. 2. The rate equation for this model is shown to be v = d[P]/dt = (kcat.[E]T[S]o/A)/[(KmBC/DA) + [S]o], where Km is the usual assembly of rate constants around EHS and A-D are functions of the form (1 + [H]/K1 + K2/[H]), in which K1 and K2 are: in A, the molecular ionization constants of ES; in B, the analogous constants of E; in C and D, apparent ionization constants composed of molecular ionization constants (of E or ES) and assemblies of rate constants. 3. As in earlier treatments of this type of reaction which involve either the assumption that the reactants and intermediate are in equilibrium or the assumption of Peller & Alberty [(1959) J. Am. Chem. Soc. 81, 5907-5914] that only EH and EHS interconvert directly, the pH-dependence of kcat. is determined only by A. 4. The pH-dependence of Km is determined in general by B-C/A-D, but when reactants and intermediate are in equilibrium, C identical to D and this expression simplifies to B/A. 5. The pH-dependence of kcat./Km, i.e. of the rate when [S]o less than Km, is not necessarily a simple bell-shaped curve characterized only by the ionization constants of B, but is a complex curve characterized by D/B-C. 6. Various situations are discussed in which the pH-dependence of kcat./Km is determined by assemblies simpler than D/B-C. The special situation in which a kcat./Km-pH profile provides the molecular pKa values of the intermediate ES complex is delineated.

摘要

考虑在“布里格斯 - 霍尔丹”条件下，E与S之间通过中间体ES进行的反应的pH依赖性，即ES存在稳态且[S]ₒ大于[E]ₜ，其中[S]ₒ是S的初始浓度，[E]ₜ是E的所有形式的总浓度。假定反应物和中间体在三种质子化状态下相互转化（E平衡于ES；EH平衡于EHS；EH₂平衡于EH₂S），但只有EHS通过不可逆反应提供产物，其速率常数为kcat。假定质子化非常快，以至于它们都处于平衡状态。2. 该模型的速率方程显示为v = d[P]/dt = (kcat·[E]ₜ[S]ₒ/A)/[(KmBC/DA) + [S]ₒ]，其中Km是围绕EHS的通常速率常数组合，A - D是形式为(1 + [H]/K₁ + K₂/[H])的函数，其中K₁和K₂为：在A中，ES的分子电离常数；在B中，E的类似常数；在C和D中，由分子电离常数（E或ES的）和速率常数组合构成的表观电离常数。3. 如同对这类反应的早期处理，要么假定反应物和中间体处于平衡状态，要么采用佩勒和阿尔伯蒂的假设[（1959年）《美国化学会志》81, 5907 - 5914]，即只有EH和EHS直接相互转化，kcat的pH依赖性仅由A决定。4. Km的pH依赖性一般由B - C/A - D决定，但当反应物和中间体处于平衡状态时，C与D相同，此表达式简化为B/A。5. kcat/Km的pH依赖性，即当[S]ₒ小于Km时的速率的pH依赖性，不一定是仅由B 的电离常数表征的简单钟形曲线，而是由D/B - C表征的复杂曲线。6. 讨论了各种情况，其中kcat/Km的pH依赖性由比D/B - C更简单的组合决定。描绘了kcat/Km - pH曲线提供中间体ES复合物的分子pKa值的特殊情况。