IMPLICATION OF HIS(68) IN THE SUBSTRATE SITE OF BACILLUS-SUBTILIS ADENYLOSUCCINATE LYASE BY MUTAGENESIS AND AFFINITY LABELING WITH 2-[(4-BROMO-2,3-DIOXOBUTYL)THIO]ADENOSINE 5'-MONOPHOSPHATE

Adenylosuccinate lyase of Bacillus subtilis is inactivated by 2-[(4-br omo-2,3-dioxobutyl)thio] adenosine 5'-monophosphate (2-BDB-TAMP) at pH 7.0. As the reagent concentration is increased, a maximum rate consta nt is approached, indicative of reversible enzyme-reagent complex form ation (K-R = 68 +/- 9 mu M) prior to irreversible modification (k(max) = 0.081 +/- 0.004 min(-1)). Complete inactivation occurs concomitant with about 1 mol of 2-BDB-[C-14]TAMP incorporated/mol of enzyme subuni t. Adenylosuccinate, or a combination of AMP and fumarate, decreases t he inactivation rate and reduces incorporation of [C-14] reagent, wher eas either AMP or fumarate alone is much less effective. These observa tions suggest that 2-BDB-TAMP attacks the adenylosuccinate binding sit e. Proteolytic digestion of inactivated enzyme, followed by purificati on of the digest by HPLC, yields the radioactive peptide Ile(62)-Ala(7 2), in which Arg(67) and His(68) are the most likely targets. Thus 2-B DB-TAMP reacts with adenylosuccinate lyase at a site distinct from the His(141) attacked by 6-BDB-TAMP (Lee, Worby, Dixon, and Colman (1997) J. Biol. Chem. 272, 458-465). Site-directed mutagenesis was used to c onstruct mutant enzymes with replacements for both Arg(67) and His(68) , and either Arg(67) or His(68). The R67M mutant enzyme has almost the same specific activity as the wild-type enzyme under standard assay c onditions, whereas the single mutant H68Q and double mutant R67M-H68Q enzymes exhibit specific activities that are decreased more than 100-f old. These results indicate that while Arg(67) and His(68) may both be in the region of the substrate site, only His(68) is important for th e catalytic activity of B. subtilis adenylosuccinate lyase. A role is proposed for His(68) as a general acid-base catalyst.