The Substrate Analog, Bromopyruvate, as Both a Substrate and Alkylating Agent for 2-Keto-3-deoxy-6-phosphogluconic Aldolase

作者：H. Paul Meloche, Mark A. Luczak, James M. Wurster · 发表于：Journal of Biological Chemistry · 年份：1972 · DOI：10.1016/s0021-9258(19)45058-8 · 被引用次数：34 · 研究领域：Folate and B Vitamins Research、Biochemical and Molecular Research、Microbial metabolism and enzyme function

Previous work has shown the substrate analog bromopyruvate to be an active site-specific alkylating agent for 2-keto-3-deoxy-6-phosphogluconic aldolase. The present work shows that [3H]bromopyruvate is detritiated by the enzyme indicating that the reagent is, in fact, a substrate. A kinetic expression derived from a model in which both catalysis and inactivation occur at the same protein site requires a constant amount of detritiation per mole of enzyme inactivated. This model was tested using the aldolase-bromopyruvate system, with the reagent over a range of concentrations which affect the inactivation rate. Over this concentration range the ratio of bromopyruvate detritiated to aldolase inactivated remained constant, as the derivation predicted, at about 50:1. In addition, excess enzyme removed only half of the tritium of (3R,S)-[3H]bromopyruvate showing the reaction to be stereospecific. Additional experiments showed that the product of reagent detritiation was (3S)-[3-3H]bromopyruvate. Thus, the aldolase activates the hydrogen in the Pro-R position of bromopyruvate. (3S)-[3-3H]Bromopyruvate was used to inactivate the aldolase. The [3-3H]carboxyketo-methyl enzyme was oxidatively decarboxylated with H2O2. The ester linkage to the enzyme was broken with hydroxylamine. The resulting tritiated glycolate was detritiated by glycolate oxidase indicating it to be of the 2R configuration. Thus the alkylation of the enzyme-carboxyl group must occur by an SN2 process. The data suppo...