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Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA
Reprint requests to: Mary F. Roberts, Merkert Chemistry Center, Boston College, Chestnut Hill, MA 02467, USA; e-mail: mary.roberts{at}bc.edu; fax: (617) 552-2705.
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 Ca2+ in modulating PLD reactions. Although the two PLDs exhibit similar substrate specificity (phosphatidylcholine preferred), sensitivity to substrate aggregation or Ca2+, 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. Ca2+, 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, Ca2+ relieves product inhibition by interactions with the phosphatidic acid (PA). A similar rate enhancement could occur with other HxKx4D-motif PLDs as well. For S. chromofuscus PLD, Ca2+ is absolutely critical for binding of the enzyme to PC vesicles and for PA activation. That the Ca2+-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.
Keywords: Phospholipase D; 31P NMR; phosphatidylcholine; phosphatidic acid; Ca2+ activation
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