Document Type

Article

Publication Date

3-1997

Abstract

The kinetics of the reaction of aspartate aminotransferase with erythro-beta-hydroxy-aspartate, in which rapid mixing is followed (upon reaching a suitable stationary state) by a very fast temperature jump, is numerically simulated. Values for rate constants are used to the extent known, otherwise estimated. It is shown that reaction steps not resolvable by rapid mixing can be resolved by subsequent chemical relaxation. Since several absorption spectra of enzyme complexes overlap, use of a pH-indicator is investigated. When the pH-indicator is coupled to the protonic dissociation of free enzyme, the fast steps are easily detected in the chemical relaxation portion of the simulation. When the pH-indicator is coupled to the protonic dissociation of the (short-lived) quinoid intermediate, protonic dissociation is easily detectable in the stopped flow phase and in the chemical relaxation phase. Such transient protonic dissociation has not been detected experimentally, but is predicted by the simulation. When natural substrates are used, the magnitude of the rate constants makes it unlikely that transient proton dissociation can be detected by stopped flow alone, but a combination of stopped flow with very fast temperature perturbation allows detection of the transient proton through use of a suitable nonbinding pH-indicator. This is demonstrated by simulation for a specific case. Finally, an alternate mechanism is introduced and distinction of its kinetics from that of the original mechanism is demonstrated.

Publication Title

Biophysical Journal

Volume

72

Issue

3

First Page

1135

Last Page

1142

DOI

http://dx.doi.org/10.1016/S0006-3495(97)78762-9

Required Publisher's Statement

Copyright 1997 Biophysical Society. The original published version of this article may be found at http://dx.doi.org/10.1016/S0006-3495(97)78762-9.

Subjects - Topical (LCSH)

Aspartate aminotransferase; Chemical kinetics; Chemical reactions

Genre/Form

articles

Type

Text

Rights

Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.

Language

English

Format

application/pdf

Included in

Mathematics Commons

COinS