NMR STRUCTURAL STUDIES OF THE TIGHT COMPLEX BETWEEN A TRIFLUOROMETHYLKETONE INHIBITOR AND THE 85-KDA HUMAN PHOSPHOLIPASE-A(2)

Arachidonyl trifluoromethyl ketone (AACOCF3) is a slow- and tight-bind ing inhibitor of the human cytosolic phospholipase A2 (cPLA2) [Street et al.(1993) Biochemistry 32,5935]. F-19 and C-13 NMR experiments have been carried out to elucidate the structure of the cPLA2.AACOCF3 comp lex. One mole of AACOCF3 per mole of enzyme is tightly bound in the ac tive site while excess molar equivalents of the inhibitor associate lo osely and nonspecifically with hydrophobic regions of the protein. Inc ubation of the cPLA2.AACOCF3 complex with a 10-fold molar excess of a structurally related inhibitor allows the slow dissociation of the enz yme-inhibitor complex to be followed with F-19 NMR. These results esta blish that the bound inhibitor is in slow exchange with the free ligan d and that inhibition of the cPLA2 by AACOCF3 is not due to irreversib le modification of the protein. AACOCF3 labeled with C-13 at the carbo nyl position was used to determine the nature of the bound inhibitor s pecies. A comparison of the C-13 NMR chemical shift value obtained fro m labeled enzyme-inhibitor complex (delta(C) 101.0 ppm) with the chemi cal shift values obtained from model compounds suggests that the enzym e-bound inhibitor species is a charged hemiketal. These results are ve ry similar to those obtained previously with alpha-chymotrypsin and a peptidyl trifluoromethyl ketone inhibitor [Liang, T.-C., & Abeles, R. H. (1987) Biochemistry 26, 7603] and, by analogy with the serine prote ases, a structural model for the cPLA2.AACOCF3 complex is proposed.