PMID:7993900
| Citation |
Li, Y, Yu, BZ, Zhu, H, Jain, MK and Tsai, MD (1994) Phospholipase A2 engineering. Structural and functional roles of the highly conserved active site residue aspartate-49. Biochemistry 33:14714-22 |
|---|---|
| Abstract |
Site-directed mutagenesis and high-resolution two-dimensional (2D) proton nuclear magnetic resonance (NMR) were used to probe the structural and functional roles of a highly conserved residue, Asp-49, in the interfacial catalysis by bovine pancreatic phospholipase A2 (PLA2, overexpressed in Escherichia coli). According to crystal structures, the side chain carboxylate of Asp-49, along with the carbonyl oxygens of Tyr-28, Gly-30, and Gly-32, and two water molecules, provides the necessary ligands for Ca2+ which is essential for the enzymatic activity. The Asp-49 of PLA2 was changed to Asn, Glu, Gln, Lys, and Ala; the resulting mutants are named D49N, D49E, D49Q, D49K, and D49A, respectively. The conformational stabilities of all five mutants are similar to that of WT as judged by guanidine hydrochloride-induced denaturation. The structural analyses by NMR indicated no global perturbations upon substitutions, although localized conformational perturbations can be observed for less conserved replacements. Direct Ca2+ binding studies showed no specific binding for D49A, D49N, D49Q, and D49K; however, D49E retains a 12-fold weaker calcium binding affinity (Kd,Ca = 23 mM). The specific activities of all five mutant enzymes decrease significantly, ranging from 5.4 x 10(2)- to 5.8 x 10(5)-fold in comparison with that of the wild-type enzyme. The observed activities of mutants require the presence of Ca2+. This demonstrates the functional importance of Asp-49 in the catalytic mechanism of PLA2, presumably by helping to bind and properly orient Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS) |
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| Keywords |
Animals; Aspartic Acid/physiology; Base Sequence; Binding Sites; Calcium/metabolism; Catalysis; Cattle; Kinetics; Models, Chemical; Molecular Sequence Data; Mutagenesis, Site-Directed; Pancreas/enzymology; Phospholipases A/chemistry; Phospholipases A/metabolism; Phospholipases A2; Protein Binding; Protein Conformation; Spectrum Analysis/methods; Structure-Activity Relationship |
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