A KINETIC INVESTIGATION OF INTERACTION OF ERYTHRO-BETA-HYDROXYASPARTIC ACID WITH ASPARTATE AMINOTRANSFERASE

Temperature-jump, stopped-flow, and spectrophotometric techniques have been used to study the kinetics of the interaction of erythro-β-hydroxyaspartic acid with aspartate aminotransferase. Eleven relaxation times were observed in a time range of <10 μsec->1 day. Experiments were monitored at 492, 460, 430, 362, 332, and 310mμ in order to determine the wavelength dependence of each step. Eight of the relaxation times are inferred to be directly related to the transamination reaction, two are probably characteristic of product decomposition, and one is believed to be associated with a nonproductive side reaction. A simple sequential reaction mechanism is proposed involving seven different intermediates, and the associated rate constants have been calculated by use of a computer analysis of the data. The spectral and structural features of some of the intermediates are inferred from the data. The proposed mechanism involves multiple conformational states of aldimine, quinoid, and ketimine structures. The quinoid intermediate has an extinction coefficient at 490μ greater than 105 m-1 cm-1. The rate-determining step is tentatively assigned to the hydrolysis or a conformational change of the ketimine. These results imply that the mechanism of enzymatic transamination involves at least 16 discrete steps. The occurrence of many reaction intermediates may be an important characteristic of enzymatic catalysis. © 1969, American Chemical Society. All rights reserved.