Abstract:
:Pancreatic phospholipase A(2) (PLA2) shows a strong preference for the binding to the anionic interface and a consequent allosteric activation. In this paper, we show that virtually all the preference is mediated through 3 (Lys-53, -56, and -120) of the 12 cationic residues of bovine pancreatic PLA2. The lysine-to-methionine substitution enhances the binding of the enzyme to the zwitterionic interface, and for the K53,56,120M triple mutant at the zwitterionic interface is comparable to that for the wild type (WT) at the anionic interface. In the isomorphous crystal structure, the backbone folding of K53,56M K120,121A and WT are virtually identical, yet a significant change in the side chains of certain residues, away from the site of substitution, mostly at the putative contact site with the interface (i-face), is discernible. Such reciprocity, also supported by the spectroscopic results for the free and bound forms of the enzyme, is expected because a distal structural change that perturbs the interfacial binding could also affect the i-face. The results show that lysine-to-methionine substitution induces a structural change that promotes the binding of PLA2 to the interface as well as the substrate binding to the enzyme at the interface. The kinetic results are consistent with a model in which the interfacial Michaelis complex exists in two forms, and the complex that undergoes the chemical step is formed by the charge compensation of Lys-53 and -56. Analysis of the incremental changes in the kinetic parameters shows that the charge compensation of Lys-53 and -56 contributes to the activation and that of Lys-120 contributes only to the structural change that promotes the stability of the Michaelis complex at the interface. The charge compensation effects on these three residues also account for the differences in the anionic interface preference of the evolutionarily divergent secreted PLA2.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Yu BZ,Poi MJ,Ramagopal UA,Jain R,Ramakumar S,Berg OG,Tsai MD,Sekar K,Jain MKdoi
10.1021/bi000740ksubject
Has Abstractpub_date
2000-10-10 00:00:00pages
12312-23issue
40eissn
0006-2960issn
1520-4995pii
bi000740kjournal_volume
39pub_type
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