Assignment of free and disulfide-bonded cysteine residues in testis angiotensin-converting enzyme: functional implications.
Human testicular angiotensin-converting enzyme (tACE) is an extracellular protein that contains seven cysteine residues. The cysteines occur in a sequential distribution that is precisely mimicked in the tACE from rabbit and mouse, and in both domains of all known species of somatic ACE. One of the cysteines in human tACE, Cys496, is present in the reduced form as shown by labeling it with 5-[[2-(iodoacetyl)amino]ethylamino]naphthalene-1-sulfonic acid, isolating the fluorescent peptide from enzymatic digests by HPLC, and analyzing its sequence by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). Thiol reagents have no significant effect on the activity of tACE, indicating that this Cys is not involved in catalysis. The other six cysteines exist as three disulfides. Mass spectral analysis of cyanogen bromide peptides has established that the cystine connectivities follow a nearest-neighbor, aabbcc, pattern i.e., Cys152-Cys158, Cys352-Cys370, and Cys538-Cys550, in which the disulfides form three small loops of five, 17, and 11 residues, respectively. Although these disulfide loops constitute less than 5% of the total sequence of the protein, they contribute to the overall structural stabilization of tACE.
Sturrock
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ED
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.
Yu
X C
XC
Wu
Z
Z
Biemann
K
K
Riordan
J F
JF
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D005453:Fluorescence; D006801:Humans; D008297:Male; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013439:Sulfhydryl Reagents / Q000378:metabolism / Q000494:pharmacology; D013737:Testis / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; C006422:1,8-I-AEDANS; D007703:Peptidyl-Dipeptidase A; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D003545:Cysteine; D003488:Cyanogen Bromide