Identification of His141 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 microM 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate (6-BDB-TAMP) at pH 7.0 and 25 degrees C. The initial inactivation rate constant exhibits nonlinear dependence on the concentration of 6-BDB-TAMP, implying there is reversible formation of enzyme-reagent complex (K(I) = 30 +/- 4 microM) prior to irreversible modification (kmax = 0.139 +/- 0.005 min(-1)). The tetrameric enzyme incorporates about 1 mol of 6-BDB-[32P]TAMP per mol of enzyme subunit concomitant with complete inactivation. Protection against inactivation and incorporation of [32P]reagent is provided by adenylosuccinate or a combination of AMP and fumarate, whereas either AMP or fumarate alone is much less effective. These observations suggest that 6-BDB-TAMP targets the adenylosuccinate-binding site. Hydrolyzed 6-BDB-TAMP is a competitive inhibitor with respect to adenylosuccinate in the catalytic reaction and also decreases the rate of inactivation by 6-BDB-TAMP. These results account for the decrease in the inactivation rate as the reaction of 6-BDB-TAMP with the enzyme proceeds. Purification by chromatography on dihydroxyboryl-agarose and high performance liquid chromatography of the tryptic digest of inactivated enzyme yields a single radioactive peptide, Thr140-Phe150, as determined by gas-phase sequencing. Modified His141 is the reaction product of 6-BDB-TAMP and adenylosuccinate lyase. We conclude that 6-BDB-TAMP functions as a reactive adenylosuccinate analog in modifying His141 in the substrate-binding site of adenylosuccinate lyase, where it may serve as a general base accepting a proton from the succinyl group during catalysis.
Lee
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TT
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
Worby
C
C
Dixon
J E
JE
Colman
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RF
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000264:Adenylosuccinate Lyase / Q000648:ultrastructure*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D016415:Sequence Alignment
D000345:Affinity Labels; D001426:Bacterial Proteins; D008024:Ligands; D000249:Adenosine Monophosphate; D006639:Histidine; C042297:6-(6-(4-bromo-2,3-dioxobutyl)thio)deaminoadenosine 5'-monophosphate; D000264:Adenylosuccinate Lyase