Northrop D B
Biochemistry. 1981 Jul 7;20(14):4056-61. doi: 10.1021/bi00517a017.
A general equation is proposed for representing the kinetic functions which govern the expression of an isotope effect on the maximal velocity of an enzyme-catalyzed reaction. The origin and form of the functions are illustrated by examining a series of enzymatic mechanisms of progressively increasing complexity. The number of functions similarly increase, reaching a limit of three, with differing thermodynamic and kinetic properties. Further expansion of mechanisms causes an orderly and predictable algebraic expansion of each function, making it possible to write out, by simple inspection, the kinetic equation describing an isotope effect expressed on the maximal velocity for any enzymatic mechanism in which the isotope perturbs a single reactive step. The functions are interactive and allow for the possibility that an isotope effect on Vmax may be independent of the rate of a second, isotopically insensitive step, be it infinitely fast or slow. This allowance leads to an uncertainty of the ability of an isotope effect to detect a rate-limiting step, and the unequal distribution of kinetic and thermodynamic properties among three functions leads to an inadequacy of the singular concept of a rate-limiting step to serve as a basis for interpreting isotope effects on enzyme-catalyzed reactions. A minimal mechanism for consideration of isotope effects is proposed in order to embrace all three functions. It consists of a single catalytic step which is isotopically sensitive and reversible, two reversible precatalytic steps, and one reversible postcatalytic step, plus steps for binding and release of substrates and products.
提出了一个通用方程，用于表示控制同位素效应在酶催化反应最大速度上表达的动力学函数。通过研究一系列复杂度逐渐增加的酶促机制，说明了这些函数的起源和形式。函数的数量同样增加，达到三个的极限，具有不同的热力学和动力学性质。机制的进一步扩展会导致每个函数进行有序且可预测的代数扩展，从而能够通过简单检查写出描述同位素效应在最大速度上表达的动力学方程，该同位素效应适用于同位素干扰单个反应步骤的任何酶促机制。这些函数是相互作用的，使得同位素效应在Vmax上可能与第二个同位素不敏感步骤的速率无关，无论该步骤是无限快还是慢。这种情况导致同位素效应检测限速步骤能力的不确定性，并且三个函数之间动力学和热力学性质的不均匀分布导致限速步骤这一单一概念不足以作为解释酶催化反应同位素效应的基础。为了涵盖所有三个函数，提出了一个考虑同位素效应的最小机制。它由一个对同位素敏感且可逆的单一催化步骤、两个可逆的催化前步骤、一个可逆的催化后步骤，以及底物和产物的结合与释放步骤组成。
