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Mechanisms of chloride modulated activity in the C-domain of angiotensin-converting enzyme
The somatic isoform of angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, primarily cleaves the hypertension-associated angiotensin-I (AngI) and bradykinin peptides, as well as a number of other physiologically relevant peptides in vitro. sACE...
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| Format: | Thesis |
| Language: | English |
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Division of Medical Biochemistry
2014
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| _version_ | 1867613710356316160 |
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| access_status_str | Open Access |
| author | Yates, Christopher John |
| author2 | Sturrock, Edward D |
| author_browse | Sturrock, Edward D Yates, Christopher John |
| author_facet | Sturrock, Edward D Yates, Christopher John |
| author_sort | Yates, Christopher John |
| collection | Thesis |
| description | The somatic isoform of angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, primarily cleaves the hypertension-associated angiotensin-I (AngI) and bradykinin peptides, as well as a number of other physiologically relevant peptides in vitro. sACE consists of two homologous and catalytically active N- and C- domains which display marked differences in substrate specificities and chloride activation. To investigate these potential mechanisms, a series of single amino acid substitution mutants (based on analysis of aligned C- and N-domain 3D structures) were generated in a soluble, minimally glycosylated C-domain construct. Evaluation of these constructs was done using AngI and the short synthetic substrates hippuryl-L-histidyl-Lleucine (HHL) and Z-phenylalanyl-L-histidyl-L-leucine (Z-FHL) under differing chloride concentrations. An isothermal titration calorimetry-based assay was developed to determine the effect of chloride concentration on enzyme thermodynamic and kinetic parameters. Chloride binding in the chloride 1 pocket of tACE was found to affect positioning of K511 and potentially alter the conformation of the active site. This would alter C-terminal substrate interactions, which were suggested to affect chloride 2 pocket ion affinity by coordinating Y520 and affect peptide bond rotation and hence substrate interactions. The analysis of the chloride 2 pocket R522Q and R522K mutations revealed a key R522-Y523 Pi-cation interaction that is stabilized via chloride coordination of R522. Substrate interactions in the S2 sub-site were shown to affect positioning of this complex as well as chloride affinity in the chloride 2 pocket. The E403-K118 salt bridge in tACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket, an interaction which is destabilized via substrate interactions within the S2 pocket which results in tighter chloride binding. This work showed that substrate composition to the C-terminal side of the scissile bond, as well as interactions of larger substrates in the S2 sub-site, moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate selective nature of chloride dependence in ACE and how this varies between the N- and C- domains. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/6885 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:40:28.535Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2014 |
| publishDateRange | 2014 |
| publishDateSort | 2014 |
| publisher | Division of Medical Biochemistry |
| publisherStr | Division of Medical Biochemistry |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/6885 Mechanisms of chloride modulated activity in the C-domain of angiotensin-converting enzyme Yates, Christopher John Sturrock, Edward D The somatic isoform of angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, primarily cleaves the hypertension-associated angiotensin-I (AngI) and bradykinin peptides, as well as a number of other physiologically relevant peptides in vitro. sACE consists of two homologous and catalytically active N- and C- domains which display marked differences in substrate specificities and chloride activation. To investigate these potential mechanisms, a series of single amino acid substitution mutants (based on analysis of aligned C- and N-domain 3D structures) were generated in a soluble, minimally glycosylated C-domain construct. Evaluation of these constructs was done using AngI and the short synthetic substrates hippuryl-L-histidyl-Lleucine (HHL) and Z-phenylalanyl-L-histidyl-L-leucine (Z-FHL) under differing chloride concentrations. An isothermal titration calorimetry-based assay was developed to determine the effect of chloride concentration on enzyme thermodynamic and kinetic parameters. Chloride binding in the chloride 1 pocket of tACE was found to affect positioning of K511 and potentially alter the conformation of the active site. This would alter C-terminal substrate interactions, which were suggested to affect chloride 2 pocket ion affinity by coordinating Y520 and affect peptide bond rotation and hence substrate interactions. The analysis of the chloride 2 pocket R522Q and R522K mutations revealed a key R522-Y523 Pi-cation interaction that is stabilized via chloride coordination of R522. Substrate interactions in the S2 sub-site were shown to affect positioning of this complex as well as chloride affinity in the chloride 2 pocket. The E403-K118 salt bridge in tACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket, an interaction which is destabilized via substrate interactions within the S2 pocket which results in tighter chloride binding. This work showed that substrate composition to the C-terminal side of the scissile bond, as well as interactions of larger substrates in the S2 sub-site, moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate selective nature of chloride dependence in ACE and how this varies between the N- and C- domains. 2014-09-02T17:13:19Z 2014-09-02T17:13:19Z 2012 Doctoral Thesis Doctoral PhD http://hdl.handle.net/11427/6885 eng application/pdf Division of Medical Biochemistry Faculty of Health Sciences University of Cape Town |
| spellingShingle | Yates, Christopher John Mechanisms of chloride modulated activity in the C-domain of angiotensin-converting enzyme |
| thesis_degree_str | Doctoral |
| title | Mechanisms of chloride modulated activity in the C-domain of angiotensin-converting enzyme |
| title_full | Mechanisms of chloride modulated activity in the C-domain of angiotensin-converting enzyme |
| title_fullStr | Mechanisms of chloride modulated activity in the C-domain of angiotensin-converting enzyme |
| title_full_unstemmed | Mechanisms of chloride modulated activity in the C-domain of angiotensin-converting enzyme |
| title_short | Mechanisms of chloride modulated activity in the C-domain of angiotensin-converting enzyme |
| title_sort | mechanisms of chloride modulated activity in the c domain of angiotensin converting enzyme |
| url | http://hdl.handle.net/11427/6885 |
| work_keys_str_mv | AT yateschristopherjohn mechanismsofchloridemodulatedactivityinthecdomainofangiotensinconvertingenzyme |