A. Kumar et al., STRUCTURE AND FUNCTION OF THE CATALYTIC SITE MUTANT ASP-99 ASN OF PHOSPHOLIPASE-A(2) - ABSENCE OF THE CONSERVED STRUCTURAL WATER, Protein science, 3(11), 1994, pp. 2082-2088
To probe the role of the Asp-99...His-48 pair in phospholipase A(2) (P
LA2) catalysis, the X-ray structure and kinetic characterization of th
e mutant Asp-99 --> Asn-99 (D99N) of bovine pancreatic PLA2 was undert
aken. 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, Biochemi
stry 30:11801-11811). The 1.9-Angstrom X-ray structure of the mutant s
howed 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 NH2
group of Asn-99 points away from His-48. In contrast, in the D102N mut
ant of the protease enzyme trypsin, the NH2 group of Asn-102 is hydrog
en bonded to His-57 resulting in the inactive tautomeric form and henc
e the loss of enzymatic activity. Although the geometry of the catalyt
ic triad in the PLA2 mutant remains the same as in the WT, we were sur
prised 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 NH2 group of Asn-99. The NH2 group now forms
a direct hydrogen bond with the carbonyl group of Ala-1.