His68 and His141 are critical contributors to the intersubunit catalytic site of adenylosuccinate lyase of Bacillus subtilis

Mutant adenylosuccinate lyases of Bacillus subtilis were prepared by site-directed mutagenesis with replacements for His(141), previously identified by affinity labeling as being in the active site [Lee, T. T., Worby, C., Dixon, J. E., and Colman, R. F. (1997) J. Biol. Chem. 272, 458-465]. Four substitutions (A, L, E, Q) yield mutant enzyme with no detectable catalytic activity, while the H141R mutant is about 10(-5) as active as the wild-type enzyme. Kinetic studies show, for the H141R enzyme, a K-m that is only 3 times that of the wild-type enzyme. Minimal activity was also observed for mutant enzymes with replacements for His(68) [Lee, T. T., Worby, C., Bao, Z.-Q., Dixon, J. E., and Colman, R. F. (1998) Biochemistry 37, 8481-8489]. Measurement of the reversible binding of radioactive adenylosuccinate by inactive mutant enzymes with substitutions at either position 68 or 141 shows that their affinities for substrate are decreased by only 10-40-fold. These results suggest that His(141), like His(68), plays an important role in catalysis, but not in substrate binding. Evidence is consistent with the hypothesis that His(141) and His(68) function, respectively, as the catalytic base and acid. Circular dichroism spectroscopy and gel filtration chromatography conducted on wild-type and all His(141) and His(68) mutants reveal that none of the mutant enzymes exhibits major structural changes and that all the enzymes are tetramers. Mixing inactive His(141) with inactive His(68) mutant enzymes leads to striking increases in catalytic activity. This complementation of mutant enzymes indicates that His(141) and His(68) come from different subunits to form the active site. A tetrameric structure of adenylosuccinate lyase was constructed by homology modeling based on the known structures in the fumarase superfamily, including argininosuccinate lyase, delta-crystallin, fumarase, and aspartase. The model suggests that each active site is constituted by residues from three subunits, and that His(141) and His(68) come from two different subunits.