IDENTIFICATION OF ESSENTIAL RESIDUES FOR THE CATALYTIC FUNCTION OF 85-KDA CYTOSOLIC PHOSPHOLIPASE A(2) - PROBING THE ROLE OF HISTIDINE, ASPARTIC-ACID, CYSTEINE, AND ARGININE

Cytosolic phospholipase A(2) (cPLA(2)) hydrolyzes the sna-acyl ester b ond of phospholipids and shows a preference for arachidonic acid-conta ining substrates. We found previously that Ser-228 is essential for en zyme activity and is likely to function as a nucleophile in the cataly tic center of the enzyme (Sharp, J. D., White, D. L., Chiou, X. G., Go odson, T., Gamboa, G. C., McClure, D., Burgett, S., Hoskins, J., Skatr ud, P. L., Sportsman, J. R., Becker, G. W., Kang, L. H., Roberts, E. F ., and Kramer, R. M. (1991) J. Biol. Chem. 266, 14850-14853). cPLA, co ntains a catalytic aspartic acid motif common to the subtilisin family of serine proteases. Substitution within this motif of Ala for Asp-54 9 completely inactivated the enzyme, and substitutions with either glu tamic acid or asparagine reduced activity 2000- and 300-fold, respecti vely. Additionally, using mutants with cysteine replaced by alanine, w e found that Cys-331 is responsible for the enzyme's sensitivity to N- ethylmaleimide, Surprisingly, substituting alanine for any of the 19 h istidines did not produce inactive enzyme, demonstrating that a classi cal serine-histidine-aspartate mechanism does not operate in this hydr olase. We found that substituting alanine or histidine for Arg-200 did produce inactive enzyme, while substituting lysine reduced activity 2 00-fold. Results obtained with the lysine mutant (R200K) and a coumari n ester substrate suggest no specific interaction between Arg-200 and the phosphoryl group of the phospholipid substrate. Arg-200, Ser-228, and Asp-549 are conserved in cPLA, from six species and also in four n onmammalian phospholipase B enzymes. Our results, supported by circula r dichroism, provide evidence that Asp-549 and Arg-200 are critical to the enzyme's function and suggest that the cPLA(2) catalytic center i s novel.