His(68) and His(141) are critical contributors to the intersubunit catalytic site of adenylosuccinate lyase of Bacillus subtilis

Mutant adenylosuccinate lyases of Bacillus subtilis were prepared by site-d irected mutagenesis with replacements for His(141), previously identified b y affinity labeling as being in the active site [Lee, T. T., Worby, C., Dix on, J. E., and Colman, R. F. (1997) J. Biol. Chem. 272, 458-465]. Four subs titutions (A, L, E, Q) yield mutant enzyme with no detectable catalytic act ivity, while the H141R mutant is about 10(-5) as active as the wild-type en zyme. Kinetic studies show, for the H141R enzyme, a K-m that is only 3 time s that of the wild-type enzyme. Minimal activity was also observed for muta nt 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]. Measu rement of the reversible binding of radioactive adenylosuccinate by inactiv e mutant enzymes with substitutions at either position 68 or 141 shows that their affinities for substrate are decreased by only 10-40-fold. These res ults suggest that His(141), like His(68), plays an important role in cataly sis, but not in substrate binding. Evidence is consistent with the hypothes is 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 no ne of the mutant enzymes exhibits major structural changes and that all the enzymes are tetramers. Mixing inactive His(141) with inactive His(68) muta nt enzymes leads to striking increases in catalytic activity. This compleme ntation of mutant enzymes indicates that His(141) and His(68) come from dif ferent subunits to form the active site. A tetrameric structure of adenylos uccinate lyase was constructed by homology modeling based on the known stru ctures in the fumarase superfamily, including argininosuccinate lyase, delt a-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.