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A new class of phospholipases A2 with lysine in place of aspartate 49. Functional consequences for calcium and substrate binding.

1984; Elsevier BV; Volume: 259; Issue: 22 Linguagem: Inglês

10.1016/s0021-9258(18)89822-2

1083-351X

John M. Maraganore, Gene Merutka, W Cho, William R. Welches, Ferenc J. Kézdy, Robert L. Heinrikson,

Enzyme function and inhibition

We report here the discovery of a new class of phospholipases Aa in which Asp-49, a residue considered to be an obligate component of the catalytic apparatus, is replaced by a lysine.Asp-49 is invariant among the more than 30 venom and pancreatic phospholipases A2 sequenced to date, and its @-carboxylate group has been shown to be a ligand for calcium in a binding site which also involves contributions from the peptide carbonyl oxygens of Tyr-28, Gly-30, and Gly-32, the so-called calcium-binding loop.The change of Asp-49 to a lysine, and other substitutions in regions heretofore thought to be invariant, including the calcium-binding loop, suggested that the new phospholipases might differ functionally with respect to calcium and/or substrate binding.Indeed, although the Lye-49 phospholipases Az show a dependence on calcium similar to that of the Asp-49 enzymes, they may be distinguished by the fact that, in the absence of phospholipid, they do not bind calcium to any measurable extent under conditions where Asp-49 enzymes bind a stoichiometric amount of calcium.Furthermore, in the absence of calcium, they show binding to single bilayer phospholipid vesicles under conditions where Asp-49 phospholipases do not bind at all.These results suggest a reversed order of addition of calcium and substrate in the formation of the ternary catalytic complex in the Lys-49 phospholipases Az.Although the mechanistic implications of these structural and functional alterations are not defined at present, it is clear that Asp-49 is not essential for phospholipase Az catalysis and that it does not participate in the enzyme-calcium-phospholipid catalytic complex.Phospholipases A2 (EC 3.1.1.4)are calcium-dependent esterases which catalyze the hydrolysis of the 2-acyl bond of 3sn-phosphoglycerides.Pancreatic juice and snake venoms are particularly rich in phospholipases A,, and enzymes from these sources have been characterized in great structural detail.Phospholipases Az are small, rigid proteins, having 6-7 disulfide bridges in a molecule approximately 125 residues