Proton Nuclear Magnetic Resonance Characterization of Phospholipase A₂ from Laticauda semifasciata

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The molecular properties of phospholipases (PLases) A₂ I and A₂ III from a sea snake, Laticauda semifasciata, have been characterized by gel-filtration, as well as proton NMR, CD, UV absorption, and fluorescence spectroscopic methods. PLase A₂ I exists as a monomer in aqueous solution in the presence or in the absence of Ca²⁺. The dissociation constants of the Ca²⁺-enzyme complexes have been determined for the two enzymes. The 270-MHz proton NMR spectra of PLases A₂ I and A₂ III have been measured, and the aromatic proton resonances of His-21 and His-48 in the active site have been assigned. By analyzing the pH dependence of the chemical shifts of the histidine proton resonances, pK_a values have been determined for His-21 and His-48 with and without Ca²⁺. The conformational transitions have been found to take place at low pH or at high temperature (at_??_65°C). Fluorescence change of PLase A₂ I upon addition of substrate analogs suggests that Trp-70 in PLase A₂ I is involved in the binding to micellar substrates. The lack of Trp-70 in PLase A₂ III is probably related to the low enzymatic activity as compared with that of PLase A₂I.