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

Abstract

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.

Intracellular PAF catabolism by PAF acetylhydrolase counteracts continual PAF synthesis

“…hydrolyze long-chain cellular phospholipids (18,19), but the substrate specificity for the type I enzyme is precise; it hydrolyzes PAF, but not slightly longer chains produced during phospholipid oxidation (19,24). PAF acetylhydrolases are particularly suited for constitutive PAF hydrolysis not only through their distinctive substrate specificity but also because, unlike most cellular phospholipase A 2 activity, they are Ca 21 -independent and do not require increased intracellular free Ca 21 to initiate hydrolysis.…”

“…The group VIII enzymes, unrelated to the group VII enzymes, are intracellular and are distinguished by their complete specificity for PAF as a substrate (24). In contrast, the class VII enzymes also use oxidatively damaged phospholipids (25,26) and protect cells from oxidative insult (27).…”

CMP-N-Acetylneuraminic Acid Synthetase from Escherichia coli K1 Is a Bifunctional Enzyme

“…As for ␣1 catalytic subunit of bovine brain PAF-AH isoform I, the side chains of Thr 103 , Leu 48 , and Leu 194 were identified to be involved in substrate recognition (42). These three residues were also conserved in the C-terminal half of CS as Thr 308 , Leu 258 , and Leu 399 (Fig.…”

The emerging roles of PAF acetylhydrolase

“…The group VIII enzymes are even more selective as they display complete specificity for hydrolysis of the acetyl residue of PAF (15). Group VII plasma and type II enzymes, however, also hydrolyze unmodified sn-2 fatty acyl residues up to 5 or 6 carbon atoms long, but this length restriction is greatly relaxed when the v-end of the sn-2 residue contains oxidized (i.e., aldehydic or carboxylic) functions (16).…”