TY - JOUR
T1 - Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase
AU - Ho, Y. S.
AU - Sheffield, P. J.
AU - Masuyama, J.
AU - Arai, H.
AU - Li, J.
AU - Aoki, J.
AU - Inoue, K.
AU - Derewenda, U.
AU - Derewenda, Z. S.
PY - 1999
Y1 - 1999
N2 - 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 α1 catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 Å 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 over-expressed and their structures were solved to 2.3 Å 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.
AB - 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 α1 catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 Å 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 over-expressed and their structures were solved to 2.3 Å 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.
KW - Enzyme kinetics
KW - Mutagenesis
KW - Phospholipase A2
KW - Serine hydrolase
KW - X-ray crystallography
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U2 - 10.1093/protein/12.8.693
DO - 10.1093/protein/12.8.693
M3 - Article
C2 - 10469831
AN - SCOPUS:0032859479
VL - 12
SP - 693
EP - 700
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
SN - 1741-0126
IS - 8
ER -