Identification of essential tyrosine and lysine residues in angiotensin converting enzyme: evidence for a single active site.
Inactivation of rabbit lung angiotensin converting enzyme (ACE) by 1-fluoro-2,4-dinitrobenzene (Dnp-F) has been shown to be due primarily to the modification of a tyrosine residue [Bünning, P., Kleeman, S.G., & Riordan, J.F. (1990) Biochemistry (preceding paper in this issue)]. Rabbit testicular ACE is also inactivated by Dnp-F. The specific site of modification has been identified by peptide mapping of tryptic digests of the Dnp-modified protein. Two principal 340-nm-absorbing peaks, not observed with protein modified in the presence of inhibitor, have been characterized. Amino acid and sequence analyses show that these peptides contain two distinct residues that have been selectively modified. The sequence of the major (greater than 90% of the total) modified peptide is YVEFTNK with the Dnp group on tyrosine. The sequence of the second, minor peptide is KVQDLQR with the Dnp group on lysine. Identical peptides were obtained from Dnp-modified rabbit lung ACE. These modified amino acids correspond to residues 200 and 118, respectively, in testicular ACE (human enzyme numbering). Both peptides are present only in the carboxy-terminal half-domain of lung ACE, corresponding to residues 776 and 694, respectively. These results indicate that the Dnp-F sensitive, catalytically functional active site is located in the "testicular" half of lung ACE.
Chen
Y N
YN
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Riordan
J F
JF
D000595:Amino Acid Sequence; D000806:Angiotensin-Converting Enzyme Inhibitors; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004139:Dinitrofluorobenzene / Q000494:pharmacology; D008168:Lung / Q000201:enzymology; D008239:Lysine; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D011817:Rabbits; D014357:Trypsin; D014443:Tyrosine
D000806:Angiotensin-Converting Enzyme Inhibitors; D010446:Peptide Fragments; D014443:Tyrosine; D004139:Dinitrofluorobenzene; D007703:Peptidyl-Dipeptidase A; D014357:Trypsin; D008239:Lysine