Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s whic h hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl, The recent solutio n of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 Angstrom resolution paved the w ay for a detailed examination of the molecular basis of substrate specifici ty in this enzyme. The crystal structure suggests that the side chains of T hr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structur es were solved to 2.3 Angstrom resolution or better by X-ray diffraction me thods. Enzyme kinetics showed that, compared with wild-type protein, all th ree mutants have higher relative activity against phospholipids with sn-2 a cyl chains longer than an acetyl, However, for each of the mutants we obser ved an unexpected and substantial reduction in the V-max of the reaction. T hese results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition sugge st that the integrity of the specificity pocket is critical for the express ion of full catalytic function, thus conferring very high substrate selecti vity on the enzyme.