Phospholipase A2 engineering. Structural and functional roles of the highly conserved active site residue aspartate-49.
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)
Li
Y
Y
Department of Chemistry, Ohio State University, Columbus 43210.
Yu
B Z
BZ
Zhu
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H
Jain
M K
MK
Tsai
M D
MD
D000818:Animals; D001224:Aspartic Acid / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism; D054467:Phospholipases A2; D011485:Protein Binding; D011487:Protein Conformation; D013057:Spectrum Analysis / Q000379:methods; D013329:Structure-Activity Relationship
D001224:Aspartic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium