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Phosphohydrolase and transphosphatidylation reactions of two Streptomyces phospholipase D enzymes: Covalent versus noncovalent catalysis
Author(s) -
Yang Hongying,
Roberts Mary F.
Publication year - 2003
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.03192503
Subject(s) - phospholipase d , phosphatidic acid , chemistry , hydrolase , stereochemistry , vesicle , enzyme , active site , phospholipase , transferase , phosphatidylcholine , hydrolysis , residue (chemistry) , biochemistry , phospholipid , membrane
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.