PROBING THE ROLES OF ACTIVE-SITE RESIDUES IN PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C FROM BACILLUS-CEREUS BY SITE-DIRECTED MUTAGENESIS

The role of amino acid residues located in the active site pocket of p hosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus ce reus [Heinz, D. W., Ryan, M., Bullock, T., & Griffith, O. H. (1995) EM BO J. 14, 3855-3863] was investigated by site-directed mutagenesis, ki netics, and crystal structure analysis. Twelve residues involved in ca talysis and substrate binding (His32, Arg69, His82, Gly83, Lys115, Glu 117, Arg163, Trp178, Asp180, Asp198, Tyr200, and Asp274) were individu ally replaced by 1-3 other amino acids, resulting in a total number of 21 mutants. Replacements in the mutants H32A, H32L, R69A, R69E, R69K, H82A, H82L, E117K, R163I, D198A, D198E, D198S, Y200S, and D274S cause d essentially complete inactivation of the enzyme. The remaining mutan ts (G83S, K115E, R163K, W178Y, D180S, Y200F, and D274N) exhibited redu ced activities up to 57% when compared with wild-type PI-PLC. Crystal structures determined at a resolution ranging from 2.0 to 2.7 Angstrom for six mutants (H32A, H32L, R163K, D198E, D274N, and D274S) showed t hat significant changes were confined to the site of the respective mu tation without perturbation of the rest of the structure. Only in muta nt D198E do the side chains of two neighboring arginine residues move across the inositol binding pocket toward the newly introduced glutami c acid. An analysis of these structure-function relationships provides new insight into the catalytic mechanism, and suggests a molecular ex planation of some of the substrate stereospecificity and inhibitor bin ding data available for this enzyme.