{"id":{"repo_id":"cape-town","oai_identifier":"oai:open.uct.ac.za:11427/6885"},"canonical_url":"https://search.dev.ndltd.org/etd/cape-town/oai:open.uct.ac.za:11427/6885","repository":{"repo_id":"cape-town","name":"University of Cape Town","base_url":"https://open.uct.ac.za/oai/request"},"display":{"title":"Mechanisms of chloride modulated activity in the C-domain of angiotensin-converting enzyme","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Yates, Christopher John"],"institution":"Division of Medical Biochemistry","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Sturrock, Edward D"],"committee_chairs":[],"committee_members":[],"year":2012,"date_issued":"2012","date_published":"2012","updated_at":"2026-07-22T22:23:35Z","subjects":[],"languages":["eng"],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/11427/6885","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Sturrock, Edward D"]},{"key":"dc:creator","label":"Author","values":["Yates, Christopher John"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2014-09-02T17:13:19Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2014-09-02T17:13:19Z"]},{"key":"dc:date.issued","label":"Date","values":["2012"]},{"key":"dc:publisher.department","label":"Dc Publisher Department","values":["Division of Medical Biochemistry"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cape Town"]},{"key":"dc:type","label":"Dc Type","values":["Doctoral Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["PhD"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["eng"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/11427/6885"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:title","label":"Title","values":["Mechanisms of chloride modulated activity in the C-domain of angiotensin-converting enzyme"]}]}],"canonical_facts":{"dc:contributor.advisor":["Sturrock, Edward D"],"dc:creator":["Yates, Christopher John"],"dc:date.accessioned":["2014-09-02T17:13:19Z"],"dc:date.available":["2014-09-02T17:13:19Z"],"dc:date.issued":["2012"],"dc:description.abstract":["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."],"dc:identifier.uri":["http://hdl.handle.net/11427/6885"],"dc:language.iso":["eng"],"dc:publisher.department":["Division of Medical Biochemistry"],"dc:publisher.institution":["University of Cape Town"],"dc:title":["Mechanisms of chloride modulated activity in the C-domain of angiotensin-converting enzyme"],"dc:type":["Doctoral Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["PhD"]},"updated_at":"2026-07-22T22:23:35Z"}