The Substrate Analog, Bromopyruvate, as a Bridging Agent for the Active Site of 2-Keto-3-deoxy-6-phosphogluconic Aldolase: CHEMICAL EVIDENCE FOR A CARBOXYLATE ADJACENT TO THE SCHIFF'S BASE-FORMING LYSINE

Abstract

Abstract The enzyme, 2-keto-3-deoxy-6-phosphogluconic aldolase is inactivated by the substrate analog, bromopyruvate. Inactivation results from either an active site carboxyl group or a cysteine being alkylated by bromopyruvate. Treatment of enzyme inactivated by carboxylate ester formation with borohydride produces the secondary amine resulting from reduction of a Schiff's base between the covalently fixed carboxyketomethyl group and the e-amino group of lysine. This is evidenced by the presence of N6-(1'-carboxy-2'-hydroxy)ethyl lysine in hydrolysates of inactivated, reduced enzyme. However, reduction of enzyme inactivated by thioether formation does not produce a secondary amine. These results then constitute chemical evidence that the carboxylate occurs adjacent to an active site lysine residue, which is the azomethine-forming lysine, while the bromopyruvate-sensitive cysteine is elsewhere in the active site environment.