The interaction of bromopyruvate with the active site of 2-keto-3-deoxygluconate-6-P aldolase of Pseudomonas saccharophila was investigated. The reagent inactivates the enzyme, exhibiting saturation kinetics and competition with pyruvate. The minimal inactivation half-time was 6 min, equivalent to a first-order rate constant of 0.115 min-1. The concentration of bromopyruvate giving the half-maximal inactivation rate Kinact was 50 mM. The Ksvalue of pyruvate as a competitive inhibitor was 0.85 mM. The enzyme asymmetrically detritiates (3 RS)-[3-3H2]bromopyruvate, forming, in water, (3S)-[3-3H,H]bromopyruvate. This stereochemistry is also exhibited by 2-keto-6-deoxygalactonate-6-P aldolase isolated from the same organism as well as the 2-keto-3-deoxygluconate-5-P aldolase of P. putida. Over a range of [3-3H]bromopyruvate concentrations affecting the inactivation rate, the ratio of nanomoles reagent catalytically turned over per unit of enzyme inactivated remained constant at 14:1, providing evidence that both catalysis and alkylation occur at the same protein site. © 1983 Plenum Publishing Corporation.