Assignment of Free and Disulfide-Bonded Cysteine Residues in Testis Angiotensin-Converting Enzyme:  Functional Implications

Abstract

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.

Identification of N-Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form

“…It is interesting to note that Asn 155 is located between cysteines 152 and 158, which form a disulfide bond (17), and it is "homologous" to Asn 131 in somatic ACE. We have suggested that specific disulfide formations in ACE could be critical in its folding process (17).…”

“…It is interesting to note that Asn 155 is located between cysteines 152 and 158, which form a disulfide bond (17), and it is "homologous" to Asn 131 in somatic ACE. We have suggested that specific disulfide formations in ACE could be critical in its folding process (17). This would be consistent with the report that ACE expressed in E. coli did not have a conformation that generated enzymatic activity (6).…”

High-Resolution Crystal Structures of Drosophila melanogaster Angiotensin-Converting Enzyme in Complex with Novel Inhibitors and Antihypertensive Drugs

“…Despite sharing ∼ 60% sequence identity with the C-domain, the N-domain of sACE has its own distinctive physicochemical and functional properties. It is thermally more stable than the C-domain, 8 more resistant to proteolysis under denaturing conditions 9 and is less dependent on chloride activation as compared with the C-domain. 10,11 Substrates, such as the hemoregulatory peptide AcSDKP (acetyl-Ser-Asp-Lys-Pro), 12 angiotensin 1-7, 13 and the enkephalin precursor Met 5 -Enk-Arg 6 -Phe 7 , 14 are specific for the N-domain hydrolysis, whereas the physiological substrates bradykinin and angiotensin I are hydrolysed with similar catalytic efficiency as the C-domain.…”

Angiotensin converting enzyme: the antigenic properties of the domain, role in alzheimer's disease and tumor progression

“…Расположение дисульфидных связей в молекуле тАПФ, идентифицированных ранее [39], было подтверждено рентгеноструктурным анализом тАПФ [15]. Анализ положения эпитопов относительно остатков цистеина, образующих дисульфидные связи в молекуле АПФ, показал, что ни один из спаренных остатков цистеина в обоих доменах не лежит в пределах выявленных антигенных детерминант (рис.…”