Effect of Asp69 and Arg310 on the pK of His68, a key catalytic residue of adenylosuccinate lyase

Adenylosuccinate lyase (ASL) of Bacillus subtilis contains three conserved histidines, His68, His89, and His141, identified by affinity labeling and site‐directed mutagenesis as critical to the intersubunit catalytic site. The pH‐Vmax profile for wild‐type ASL is bell‐shaped (pK1 = 6.74 and pK2 = 8.28). Only the alkaline side changes with temperature, characteristic of histidine pKs. To identify determinants of pK2 in the enzyme‐substrate complex, we replaced residues at two positions close to His68 (but not to His89 or His141) in the structure. Compared with the specific activity of 1.75 μmol adenylosuccinate reacting/min/mg of wild‐type enzyme at pH 7.0, mutant enzymes D69E, D69N, R310Q, and R310K exhibit specific activities of 0.40, 0.04, 0.00083, and 0.10, respectively. While D69E has a Km for adenylosuccinate similar to that of wild‐type ASL, D69N and R310K exhibit modest increases in Km, and R310Q has an 11‐fold increase in Km. The mutant enzymes show no significant change in molecular weight or secondary structure. The major change is in the pH‐Vmax profile: pK2 is 8.48 for the D69E mutant and is decreased to 7.83 in D69N, suggesting a proximal negative charge is needed to maintain the high pK of 8.28 observed for wild‐type enzyme and attributed to His68. Similarly, R310Q exhibits a decrease in its pK2 (7.33), whereas R310K shows little change in pK2 (8.24). These results suggest that Asp69 interacts with His68, that Arg310 interacts with and orients the β‐carboxylate of Asp69, and that His68 must be protonated for ASL to be active.