Essential residues in lysolecithin:lysolecithin acyltransferase from rabbit lung: assessment by chemical modification.
The inhibition of lysolecithin:lysolecithin acyltransferase by several specific reagents was studied. Diisopropyl fluorophosphate (DFP) completely inhibited both activities at a concentration of 4 mM. Activity was not protected by substrate and the enzyme showed a change in circular dichroism spectrum upon treatment with inhibitor. Phenylmethanesulfonyl fluoride, another serine-specific reagent, did not inhibit either hydrolysis or transacylation. Therefore, we suggest that DFP does not modify an active serine in the catalytic site. p-Hydroxymercury benzoate and N-ethylmaleimide (NEM) abolished both activities of the enzyme. The presence of substrate partially protected against inactivation. Far-uv CD spectrum of NEM-modified enzyme revealed no changes in protein structure. The existence of two classes of essential cysteine residues was deduced from kinetics of NEM inactivation. Both classes differ in NEM reactivity and also in their participation in the catalytic mechanism. A tyrosine-specific reagent, tetranitromethane, also inhibited hydrolysis and transacylation, following first-order kinetics. The partial protection by substrate suggested the possible existence of essential tyrosines near the active site. At pH 5.0 N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline inactivated hydrolysis but not transacylation. However, both of them remained unchanged at pH 6.5. The substrate prevented the loss of hydrolytic ability. Therefore, a carboxyl residue participating just in the catalytic mechanism of hydrolysis is proposed.
Pérez-Gil
J
J
Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias Químicas, Universidad Complutense, Madrid, Spain.
Martín
J J
JJ
Acebal
C
C
Arche
R
R
D008243:1-Acylglycerophosphocholine O-Acyltransferase / Q000037:antagonists & inhibitors*; D000217:Acyltransferases / Q000037:antagonists & inhibitors*; D000818:Animals; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011804:Quinolines / Q000494:pharmacology; D011817:Rabbits; D013774:Tetranitromethane / Q000494:pharmacology
D006902:Hydroxymercuribenzoates; D011804:Quinolines; D007531:Isoflurophate; C030601:4-hydroxymercuribenzoate; D010664:Phenylmethylsulfonyl Fluoride; C000470:EEDQ; D000217:Acyltransferases; D008243:1-Acylglycerophosphocholine O-Acyltransferase; D013774:Tetranitromethane