catalytic sequence HxKxxxxD (HKD), where H, K, and D are the amino acids histidine (H), lysine (K), aspartic acid (D), while x represents nonconservative amino acids. These two HKD motifs confer hydrolytic activity to PLD, and are critical for its enzymatic activity both in vitro and in vivo. Hydrolysis of the phosphodiester bond occurs when these HKD sequences are in the correct proximity. Human proteins containing this motif include: PGS1, PLD1, PLD2, PLD3, PLD4, PLD5 PC-hydrolyzing PLD is a homologue of cardiolipin synthase, phosphatidylserine synthase, bacterial PLDs, and viral proteins. Each of these appears to possess a domain duplication which is apparent by the presence of two HKD motifs containing well-conserved histidine, lysine, and asparagine residues which may contribute to the active site aspartic acid. An Escherichia coli endonuclease (nuc) and similar proteins appear to be PLD homologues but possess only one of these motifs. PLD genes additionally encode highly conserved regulatory domains: the phox consensus sequence (PX), the pleckstrin homology domain (PH), and a binding site for phosphatidylinositol 4,5-bisphosphate (PIP2). Mechanism of catalysis PLD-catalyzed hydrolysis has been proposed to occur in two stages via a "ping-pong" mechanism. In this scheme, the histidine residues of each HKD motif successively attack the phospholipid substrate. Functioning as nucleophiles, the constituent imidazole moieties of the histidines form transient covalent bonds with the phospholipid, producing a short-lived intermediate that can be easily hydrolyzed by water in a subsequent step. Mechanism of activation Substrate presentation For mammalian PLD2, the molecular basis of activation is substrate presentation. The enzyme resides inactive in lipid micro-domains rich in sphingomyelin and depleted of PC substrate. Increased PIP2 or a decrease in cholesterol causes the enzyme to translocate to PIP2 micro domains near its substrate PC. Hence PLD can is primarily activated by localization within the plasma membrane rather than a protein conformational change. Disruption of lipid domains by anesthetics. or mechanical force. The protein may also undergo a conformational change upon PIP2 binding, but this has not been shown experimentally and would constitute a mechanism of activation distinct from substrate presentation. Isoforms Two major isoforms of phospholipase D has been identified in mammalian cells: PLD1 and PLD2 (53% sequence homology), each encoded by distinct genes. PLD activity appears to be present in most cell types, with the possible exceptions of peripheral leukocytes and other lymphocytes. Both PLD isoforms require PIP2 as a cofactor for activity. PLD1 and PLD2 exhibit