Proton nuclear magnetic resonance characterization of phospholipase A2 from Laticauda semifasciata.
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, pKa 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 approximately 65 degrees 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 A2 I.
D000818:Animals; D002118:Calcium; D002942:Circular Dichroism; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D012911:Snakes; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D006639:Histidine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium