Implication of His68 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-bromo-2,3-dioxobutyl)thio]adenosine 5'-monophosphate (2-BDB-TAMP) at pH 7.0. As the reagent concentration is increased, a maximum rate constant is approached, indicative of reversible enzyme-reagent complex formation (KR = 68 +/- 9 microM) prior to irreversible modification (kmax = 0.081 +/- 0.004 min-1). Complete inactivation occurs concomitant with about 1 mol of 2-BDB-[14C]TAMP incorporated/mol of enzyme subunit. Adenylosuccinate, or a combination of AMP and fumarate, decreases the inactivation rate and reduces incorporation of [14C] reagent, whereas either AMP or fumarate alone is much less effective. These observations suggest that 2-BDB-TAMP attacks the adenylosuccinate binding site. Proteolytic digestion of inactivated enzyme, followed by purification of the digest by HPLC, yields the radioactive peptide Ile62-Ala72, in which Arg67 and His68 are the most likely targets. Thus 2-BDB-TAMP reacts with adenylosuccinate lyase at a site distinct from the His141 attacked by 6-BDB-TAMP (Lee, Worby, Dixon, and Colman (1997) J. Biol. Chem. 272, 458-465). Site-directed mutagenesis was used to construct mutant enzymes with replacements for both Arg67 and His68, and either Arg67 or His68. The R67M mutant enzyme has almost the same specific activity as the wild-type enzyme under standard assay conditions, whereas the single mutant H68Q and double mutant R67M-H68Q enzymes exhibit specific activities that are decreased more than 100-fold. These results indicate that while Arg67 and His68 may both be in the region of the substrate site, only His68 is important for the catalytic activity of B. subtilis adenylosuccinate lyase. A role is proposed for His68 as a general acid-base catalyst.
Lee
T T
TT
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
Worby
C
C
Bao
Z Q
ZQ
Dixon
J E
JE
Colman
R F
RF
D000264:Adenylosuccinate Lyase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites / Q000187:drug effects; D002250:Carbon Radioisotopes; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D013379:Substrate Specificity; D013873:Thionucleotides / Q000378:metabolism* / Q000494:pharmacology
D000345:Affinity Labels; D002250:Carbon Radioisotopes; D013873:Thionucleotides; C064134:2-((4-bromo-2,3-dioxobutyl)thio)-adenosine 3'5'-cyclic monophosphate; D006639:Histidine; D001120:Arginine; D000242:Cyclic AMP; D000264:Adenylosuccinate Lyase