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Protein Science, Vol 3, Issue 11 2082-2088, Copyright © 1994 by Cold Spring Harbor Laboratory Press

ARTICLE

Structure and function of the catalytic site mutant Asp 99 Asn of phospholipase A(2): Absence of the conserved structural water

A. KUMAR, C. SEKHARUDU, B. RAMAKRISHNAN, C. M. DUPUREUR, H. ZHU, M. D. TSAI and M. SUNDARALINGAM
Departments of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210

To probe the role of the Asp-99...His-48 pair in phospholipase A(2) (PLA2) catalysis, the X-ray structure and kinetic characterization of the mutant Asp-99->Asn-99 (D99N) of bovine pancreatic PLA2 was undertaken. Crystals of D99N belong to the trigonal space group P3(1)21 and were isomorphous to the wild type (WT) (Noel JP et al., 1991, Biochemistry 30:11801-11811). The 1.9-A X-ray structure of the mutant showed that the carbonyl group of Asn-99 side chain is hydrogen bonded to His-48 in the same way as that of Asp-99 in the WT, thus retaining the tautomeric form of His-48 and the function of the enzyme. The NH(2) group of Asn-99 points away from His-48. In contrast, in the D102N mutant of the protease enzyme trypsin, the NH(2) group of Asn-102 is hydrogen bonded to His-57 resulting in the inactive tautomeric form and hence the loss of enzymatic activity. Although the geometry of the catalytic triad in the PLA2 mutant remains the same as in the WT, we were surprised that the conserved structural water, linking the catalytic site with the ammonium group of Ala-1 of the interfacial site, was ejected by the proximity of the NH(2) group of Asn-99. The NH(2) group now forms a direct hydrogen bond with the carbonyl group of Ala-1.
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