Short-lived intermediates in aspartate aminotransferase systems.
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.
Czerlinski
G
G
Department of Biology, Western Washington University, Bellingham 98225, USA. ghc@henson.cc.wwu.edu
Levin
R
R
Ypma
T
T
D001219:Aspartate Aminotransferases / Q000737:chemistry* / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D011485:Protein Binding; D013816:Thermodynamics
C011274:3-hydroxyaspartic acid; D001224:Aspartic Acid; D001219:Aspartate Aminotransferases