Aluminum fluoride inhibition of cabbage phospholipase D by a phosphate-mimicking mechanism

Aluminum fluoride (AlF4-) inhibited phospholipase D (PLD) purified from cab bage in both PIP2-dependent and PIP2-independent assays, consistent with it s previously observed effect on mammalian PLD, The possibility that AlF4- m ay exert this effect through its known phosphate-mimicking property,vas exa mined. Inorganic phosphate, as well as two phosphate analogs, beryllium flu oride and orthovanadate, also inhibited cabbage PLD, Enzyme kinetic studies confirmed that PLD followed Hill kinetics, characteristic for allosteric e nzymes, with an apparent Hill coefficient (n(app)) of 3.8, indicating posit ive cooperativity among multiple substrate-binding sites and suggesting pos sible functional oligomerization of the enzyme. AlF4- modification of PLD k inetics was consistent with a competitive mode of enzyme inhibition. It is therefore proposed that AlF4-, and other phosphate analogs, inhibits plant PLD by competing with a substrate phosphate group for a substrate-binding s ite, thereby preventing the formation of an enzyme-phosphatidyl intermediat e. This may be a conserved feature of PLD superfamily enzymes, (C) 1999 Fed eration of European Biochemical Societies.