Effect of Asp 69 and Arg 310 on the pK of His 68 , a key catalytic residue of adenylosuccinate lyase

Adenylosuccinate lyase (ASL) of Bacillus subtilis contains three conserved histidines, His 68 , His 89 , and His 141 , identified by affinity labeling and site‐directed mutagenesis as critical to the intersubunit catalytic site. The pH‐V max profile for wild‐type ASL is bell‐shaped (p K 1 = 6.74 and p K 2 = 8.28). Only the alkaline side changes with temperature, characteristic of histidine p K s. To identify determinants of p K 2 in the enzyme‐substrate complex, we replaced residues at two positions close to His 68 (but not to His 89 or His 141 ) in the structure. Compared with the specific activity of 1.75 μ mol 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 K m for adenylosuccinate similar to that of wild‐type ASL, D69N and R310K exhibit modest increases in K m , and R310Q has an 11‐fold increase in K m . The mutant enzymes show no significant change in molecular weight or secondary structure. The major change is in the pH‐V max profile: p K 2 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 p K of 8.28 observed for wild‐type enzyme and attributed to His 68 . Similarly, R310Q exhibits a decrease in its p K 2 (7.33), whereas R310K shows little change in p K 2 (8.24). These results suggest that Asp 69 interacts with His 68 , that Arg 310 interacts with and orients the β ‐carboxylate of Asp 69 , and that His 68 must be protonated for ASL to be active.