Protein Engineering, Vol. 12, No. 6, 497-503,
June 1999
© 1999 Oxford University Press
Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K
Department of Enzymology and Protein Engineering (CBLE, Institute of Biomembranes), Faculty of Chemistry, Utrecht University, PO Box 80.054, 3508 TB Utrecht, The Netherlands 1 The paper is dedicated to Professor Bert Verheij who died in an accident on August 1, 1998
The catalytic contribution of His48 in the active site of porcine pancreatic phospholipase A2 was examined using site-directed mutagenesis. Replacement of His48 by lysine (H48K) gives rise to a protein having a distorted lipid binding pocket. Activity of this variant drops below the detection limit which is 107-fold lower than that of the wild-type enzyme. On the other hand, the presence of glutamine (H48Q) or asparagine (H48N) at this position does not affect the structural integrity of the enzyme as can be derived from 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 105. Residual activity is totally lost after addition of a competitive inhibitor. We conclude that proper lipid binding on its own accelerates ester bond hydrolysis by a factor of 102. With the selected variants, we were also able to dissect the contribution of the hydrogen bond between Asp99 and His48 on conformational stability, being 5.2 kJ/mol. Another hydrogen bond with His48 is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycol interacts with the enzyme. Its contribution to binding of the inhibitor in the presence of an interface was found to be 5.7 kJ/mol.
Keywords: active site histidine/conformational stability/inhibitor binding/porcine pancreatic phospholipase A2/site-directed mutagenesis
2 To whom correspondence should be addressed
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