IDENTIFICATION OF HIS(141) IN THE ACTIVE-SITE OF BACILLUS-SUBTILIS ADENYLOSUCCINATE LYASE BY AFFINITY LABELING WITH 6-(4-BROMO-2,3-DIOXOBUTYL)THIOADENOSINE 5'-MONOPHOSPHATE

Adenylosuccinate lyase of Bacillus subtilis is inactivated by 25-400 m u M 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate (6-BDB-TA MP) at pH 7.0 and 25 degrees C. The initial inactivation rate constant exhibits nonlinear dependence on the concentration of 6-BDB-TAMP, imp lying there is reversible formation of enzyme reagent complex (K-I = 3 0 +/- 4 mu M) prior to irreversible modification (k(max) = 0.139 +/- 0 .005 min(-1)). The tetrameric enzyme incorporates about 1 mol of 6-BDB -[P-32]TAMP per mol of enzyme subunit concomitant with complete inacti vation. Protection against inactivation and incorporation of [P-32]rea gent is provided by adenylosuccinate or a combination of AMP and fumar ate, whereas either AMP or fumarate alone is much less effective. Thes e observations suggest that 6-BDB-TAMP targets the adenylosuccinate-bi nding site. Hydrolyzed 6-BDB-TAMP is a competitive inhibitor with resp ect to adenylosuccinate in the catalytic reaction and also decreases t he rate of inactivation by 6-BDB-TAMP. These results account for the d ecrease in the inactivation rate as the reaction of 6-BDB-TAMP with th e enzyme proceeds. Purification by chromatography on dihydroxyboryl-ag arose and high performance liquid chromatography of the tryptic digest of inactivated enzyme yields a single radioactive peptide, Thr(140)-P he(150), as determined by gas-phase sequencing. Modified His(141) is t he reaction product of 6-BDB-TAMP and adenylosuccinate lyase, We concl ude that 6-BDB-TAMP functions as a reactive adenylosuccinate analog in modifying His(141) in the substrate-binding site of adenylosuccinate lyase, where it may serve as a general base accepting a proton from th e succinyl group during catalysis.