NMR structural studies of the tight complex between a trifluoromethyl ketone inhibitor and the 85-kDa human phospholipase A2

Abstract

Arachidonyl trifluoromethyl ketone (AACOCF3) is a slow- and tight-binding inhibitor of the human cytosolic phospholipase A2 (cPLA2) [Street et al. (1993) Biochemistry 32, 5935]. 19F and 13C NMR experiments have been carried out to elucidate the structure of the cPLA2.AACOCF3 complex. One mole of AACOCF3 per mole of enzyme is tightly bound in the active site while excess molar equivalents of the inhibitor associate loosely and nonspecifically with hydrophobic regions of the protein. Incubation of the cPLA2.AACOCF3 complex with a 10-fold molar excess of a structurally related inhibitor allows the slow dissociation of the enzyme-inhibitor complex to be followed with 19F NMR. These results establish that the bound inhibitor is in slow exchange with the free ligand and that inhibition of the cPLA2 by AACOCF3 is not due to irreversible modification of the protein. AACOCF3 labeled with 13C at the carbonyl position was used to determine the nature of the bound inhibitor species. A comparison of the 13C NMR chemical shift value obtained from labeled enzyme-inhibitor complex (delta c 101.0 ppm) with the chemical shift values obtained from model compounds suggests that the enzyme-bound inhibitor species is a charged hemiketal. These results are very 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 proteases, a structural model for the cPLA2.AACOCF3 complex is proposed.

Purification and Characterization of 1-O-Acylceramide Synthase, a Novel Phospholipase A2 with Transacylase Activity

“…(17). Additionally, it was reported that AACOCF 3 is both a slow and tight binding inhibitor of cPLA 2 that may form a hemiketal enzyme-inhibitor complex (16,18). The reaction mechanism of cPLA 2 thus might be similar to that of serine protease, esterase, or lipase (15)(16)(17)(18).…”

Inhibitors of Brain Phospholipase A2Activity: Their Neuropharmacological Effects and Therapeutic Importance for the Treatment of Neurologic Disorders

“…NMR studies show that the carbon chain of AACOCF 3 binds in a hydrophobic pocket and the carbonyl group of AACOCF 3 forms a covalent bond with the serine 228 in the active site, generating a charged hemiketal oxoanion that interacts with a positively charged group of the enzyme Trimble et al, 1993). AACOCF 3 is a 500-fold more potent inhibitor of cPLA 2 than sPLA 2 (Trimble et al, 1993), indicating that it is a more selective inhibitor of cPLA 2 than sPLA 2 . This inhibitor also blocks cyclooxygenase activity (Riendeau et al, 1994).…”

Activation, Inhibition, and Regiospecificity of the Lysophospholipase Activity of the 85-kDa Group IV Cytosolic Phospholipase A2

“…Transfer of the fatty acid from both diacyl-and lysophospholipid substrates to acceptors other than water initially suggested the existence of such an acyl-enzyme intermediate (10,11). Further corroboration came when NMR studies indicated the formation of a hemiketal between arachidonyl trifluoromethyl ketone (a potent inhibitor of both activities) and an enzyme hydroxyl group (24). Finally, a serine residue (Ser-228) was identified to be essential for the PLA 2 activity and was also shown to be the site of enzyme acylation (15,25).…”