Phosphohydrolase and transphosphatidylation reactions of two Streptomyces phospholipase D enzymes: covalent versus noncovalent catalysis.

Abstract

A kinetic comparison of the hydrolase and transferase activities of two bacterial phospholipase D (PLD) enzymes with little sequence homology provides insights into mechanistic differences and also the more general role of Ca(2+) in modulating PLD reactions. Although the two PLDs exhibit similar substrate specificity (phosphatidylcholine preferred), sensitivity to substrate aggregation or Ca(2+), and pH optima are quite distinct. Streptomyces sp. PMF PLD, a member of the PLD superfamily, generates both hydrolase and transferase products in parallel, consistent with a mechanism that proceeds through a covalent phosphatidylhistidyl intermediate where the rate-limiting step is formation of the covalent intermediate. For Streptomyces chromofuscus PLD, the two reactions exhibit different pH profiles, a result consistent with a mechanism likely to involve direct attack of water or an alcohol on the phosphorus. Ca(2+), not required for monomer or micelle hydrolysis, can activate both PLDs for hydrolysis of PC unilamellar vesicles. In the case of Streptomyces sp. PMF PLD, Ca(2+) relieves product inhibition by interactions with the phosphatidic acid (PA). A similar rate enhancement could occur with other HxKx(4)D-motif PLDs as well. For S. chromofuscus PLD, Ca(2+) is absolutely critical for binding of the enzyme to PC vesicles and for PA activation. That the Ca(2+)-PA activation involves a discreet site on the protein is suggested by the observation that the identity of the C-terminal residue in S. chromofuscus PLD can modulate the extent of product activation.