Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase.
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.
Ho
Y S
YS
Department of Molecular Physiology and Biological Physics, University of Virginia, Health Sciences Center, P.O. Box 10011, Charlottesville, VA 22906-0011, USA.
Sheffield
P J
PJ
Masuyama
J
J
Arai
H
H
Li
J
J
Aoki
J
J
Inoue
K
K
Derewenda
U
U
Derewenda
Z S
ZS
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D008969:Molecular Sequence Data; D009154:Mutation; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010972:Platelet Activating Factor / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D010972:Platelet Activating Factor; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase