Essential residues in angiotensin converting enzyme: modification with 1-fluoro-2,4-dinitrobenzene.
The peptidase and esterase activities of rabbit pulmonary angiotensin converting enzyme (ACE) are rapidly abolished on reaction with 1-fluoro-2,4-dinitrobenzene (Dnp-F). Inactivation follows first-order kinetics with respect to the reagent and is accompanied by stoichiometric incorporation of 3,5-[3H]Dnp, indicating that the effect is due to a specific modification of the enzyme. Thin-layer chromatography of an acid hydrolysate of the modified enzyme indicates that most of the radioactive label is present as O-Dnp-tyrosine (65 to greater than 95%) and the rest as N epsilon-Dnp-lysine. The pH dependence of the reaction is consistent with modification of either tyrosine or lysine. The presence of a competitive inhibitor effectively protects the enzyme against inactivation by Dnp-F. Acetylation of ACE with N-acetylimidazole also protects the enzyme against modification with Dnp-F. The results indicate the presence of catalytically essential tyrosine and lysine residues at the active site of ACE.
Bünning
P
P
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Kleemann
S G
SG
Riordan
J F
JF
D000806:Angiotensin-Converting Enzyme Inhibitors; D000818:Animals; D001665:Binding Sites; D004139:Dinitrofluorobenzene / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000806:Angiotensin-Converting Enzyme Inhibitors; D007093:Imidazoles; D013439:Sulfhydryl Reagents; D004139:Dinitrofluorobenzene; D007703:Peptidyl-Dipeptidase A; C012160:N-acetylimidazole