The Application of Transient-State Kinetic Isotope Effects to the Resolution of Mechanisms of Enzyme-Catalyzed Reactions

Abstract

Much of our understanding of the mechanisms of enzyme-catalyzed reactions is based on steady-state kinetic studies. Experimentally, this approach depends solely on the measurement of rates of free product appearance (d[P]/dt), a mechanistically and mathematically complex entity. Despite the ambiguity of this observed parameter, the method's success is due in part to the elaborate rigorously derived algebraic theory on which it is based. Transient-state kinetics, on the other hand, despite its ability to observe the formation of intermediate steps in real time, has contributed relatively little to the subject due in, some measure, to the lack of such a solid mathematical basis. Here we discuss the current state of existing transient-state theory and the difficulties in its realistic application to experimental data. We describe a basic analytic theory of transient-state kinetic isotope effects in the form of three novel fundamental rules. These rules are adequate to define an extended mechanism, locating the isotope-sensitive step and identifying missing steps from experimental data. We demonstrate the application of these rules to resolved component time courses of the phenylalanine dehydrogenase reaction, extending the previously known reaction by one new prehydride transfer step and two new post hydride transfer steps. We conclude with an assessment of future directions in this area.

Evolution of Enzyme Kinetic Mechanisms

“…Transient-state kinetics deals with very rapid reactions. The reaction mechanisms are directly related to the structure of the enzyme (Alberty 2010a ; Fisher 2013 ). Steady-state enzyme kinetics are based on the assumption that the steps in the catalytic mechanism follow steady-state kinetics, with all the state variables remaining constant, despite exposure to continuous changes (Martin 2011 ).…”

Kinetic Spectrophotometric Method for the 1,4-Diionic Organophosphorus Formation in the Presence of Meldrum′s Acid: Stopped-Flow Approach

“…In addition, the kinetics of these reactions has been published recently [29,30]. Numerous kinetic studies have been reported by the stopped-flow method [31,32]. In our previous works, the applications of the stopped-flow technique in kinetic and mechanistic studies have been reported for some organic synthesis reactions as cyclic crystalline phosphorous and pyrrole phosphorous ylides [33,34].…”