Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K

The catalytic contribution of His48 in the active site of porcine pancreati c phospholipase A2 was examined using site-directed mutagenesis, Replacemen t of His48 by lysine (H48K) gives rise to a protein having a distorted lipi d binding pocket. Activity of this variant drops below the detection limit which is 10(7)-fold lower than that of the wild-type enzyme. On the other h and, the presence of glutamine (H48Q) or asparagine (H48N) at this position does not affect the structural integrity of the enzyme as can be derived f rom the preserved lipid binding properties of these variants. However, the substitutions H48Q and H48N strongly reduce the turnover number, i.e. by a factor of 10(5). Residual activity is totally lost after addition of a comp etitive inhibitor. We conclude that proper lipid binding on its own acceler ates ester bond hydrolysis by a factor of 10(2). With the selected variants , we were also able to dissect the contribution of the hydrogen bond betwee n Asp99 and His48 on conformational stability, being 5.2 kJ/mol, Another hy drogen bond with His48 is formed when the competitive inhibitor (R)-2-dodec anoylamino-hexanol-1-phosphoglycol interacts with the enzyme. Its contribut ion to binding of the inhibitor in the presence of an interface was found t o be 5.7 kJ/mol.