Essential histidine residues in lysolecithin:lysolecithin acetyltransferase from rabbit lung.
Both activities of rabbit lung lysolecithin:lysolecithin acyltransferase (EC 3.1.1.5), hydrolysis and transacylation, are inactivated by diethylpyrocarbonate. The reaction follows pseudo-first-order kinetics, and second-order rate constants of 1.17 mM-1min-1 for hydrolysis and 0.56 mM-1 min-1 for transacylation were obtained at pH 6.5 and 37 degrees C. The rate of inactivation is dependent on pH, showing the involvement of a group with a pK of 6.5. The difference spectra showed an increase in absorbance at 242 nm, indicating the modification of histidine residues. The activity lost by diethylpyrocarbonate modification can be partially recovered by hydroxylamine treatment. The statistical analysis of residual fractional activity versus the number of modified histidine residues leads to the conclusion that two histidine residues are essential for the hydrolytic activity, whereas transacylation activity depends on only one essential histidine. The substrate and substrate analogs protected the enzyme against inactivation by diethylpyrocarbonate, suggesting that the essential residues are located at or near the active site of the enzyme.
Pérez-Gil
J
J
Departamento de Bioquímica y Biologia Molecular I, Facultad de Ciencias Químicas, Universidad Complutense, Madrid, Spain.
Martin
J
J
Acebal
C
C
Arche
R
R
D008243:1-Acylglycerophosphocholine O-Acyltransferase / Q000378:metabolism*; D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet
D006639:Histidine; D000217:Acyltransferases; D008243:1-Acylglycerophosphocholine O-Acyltransferase; D004047:Diethyl Pyrocarbonate