Molecules 2013, 18(7), 8230-8242; doi:10.3390/molecules18078230
Review

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

Department of Biochemistry and Molecular Biology, University of Kansas School of Medicine, 3901 Rainbow Blvd, Kansas City, KS 66160, USA
Received: 12 May 2013; in revised form: 19 June 2013 / Accepted: 1 July 2013 / Published: 12 July 2013
(This article belongs to the Special Issue Isotope Effects)
PDF Full-text Download PDF Full-Text [1113 KB, uploaded 12 July 2013 14:09 CEST]
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.
Keywords: enzyme mechanism; isotope effects; isotope sensitive step; stopped-flow; quench flow

Article Statistics

Load and display the download statistics.

Citations to this Article

Cite This Article

MDPI and ACS Style

Fisher, H.F. The Application of Transient-State Kinetic Isotope Effects to the Resolution of Mechanisms of Enzyme-Catalyzed Reactions. Molecules 2013, 18, 8230-8242.

AMA Style

Fisher HF. The Application of Transient-State Kinetic Isotope Effects to the Resolution of Mechanisms of Enzyme-Catalyzed Reactions. Molecules. 2013; 18(7):8230-8242.

Chicago/Turabian Style

Fisher, Harvey F. 2013. "The Application of Transient-State Kinetic Isotope Effects to the Resolution of Mechanisms of Enzyme-Catalyzed Reactions." Molecules 18, no. 7: 8230-8242.

Molecules EISSN 1420-3049 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert