A Molecular Basis for the C-Domain Selectivity of Angiotensin-Converting Enzyme

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dc.contributor.advisorSturrock, Edward Den_ZA
dc.contributor.authorKroger, Wen_ZA
dc.date.accessioned2014-07-28T14:55:03Z
dc.date.available2014-07-28T14:55:03Z
dc.date.issued2009en_ZA
dc.description.abstractAngiotensin-Converting Enzyme (ACE) plays an essential role in blood pressure regulationand ACE inhibitors are widely used to treat cardiovascular disease. Two isoforms exist,somatic ACE (sACE) consisting of two homologous domains, N- and C-domain, and testisACE (tACE), corresponding to the C-domain of sACE. Despite a high degree of sequenceidentity, these two domains display marked differences in substrate and inhibitor specificity.Furthermore, the C-domain of ACE has been implicated to play a dominant role in bloodpressure control. It has therefore been suggested that development of ACE inhibitortreatments that selectively block the C-domain will result in decreased side-effects comparedto current therapies. Analysis of three-dimensional structures of tACE in complex withdomain-specific inhibitors has enabled the identification of key active-site residues potentiallyplaying a role in domain selectivity. To investigate the contribution of such residues, a seriesof C-domain mutants was generated containing single and multiple N-domain active-sitesubstitutions. These constructs were used to characterise specific interactions using domainselectiveinhibitors and fluorogenic peptides. Mutants tested with the fluorogenic peptidesdisplayed minimal, if any, acquisition of N-domain-like catalytic properties. Of the singlemutations, S2 (F391Y, tACE numbering) and S1 (V518T) pocket substitutions caused thelargest decreases in affinity for the C-selective phosphinic inhibitor RXPA380 (34-fold) andketo-ACE derivatives (14-26 fold), respectively. The V379S mutation caused an unexpectedincrease in affinity (2-10 fold) for C-selective inhibitors containing a P2’ Trp that could beexplained by the formation of a water-mediated hydrogen bond interaction resulting fromrearrangement of inhibitor and protein side-chains within the S2’ pocket. Multiple mutantscontaining an N-domain-like S2’ pocket combined with the S2 F391Y substitution (S2’F)caused the most notable shift in Ki from that of tACE for the highly selective phosphinicinhibitors, RXPA380 (Ki’s tACE = 69 nM; S2’F = 5300 nM) and N-specific RXP407 (Ki’stACE = 2800 nM; S2’F Ki = 16.1 nM). This work identifies key residues contributing to thedomain selectivity of ACE, and highlights the complex combination of effects involved in thisphenomenon. Furthermore, it provides useful insight for the further design of domainselectiveinhibitors.en_ZA
dc.identifier.apacitationKroger, W. (2009). <i>A Molecular Basis for the C-Domain Selectivity of Angiotensin-Converting Enzyme</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry. Retrieved from http://hdl.handle.net/11427/3135en_ZA
dc.identifier.chicagocitationKroger, W. <i>"A Molecular Basis for the C-Domain Selectivity of Angiotensin-Converting Enzyme."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry, 2009. http://hdl.handle.net/11427/3135en_ZA
dc.identifier.citationKroger, W. 2009. A Molecular Basis for the C-Domain Selectivity of Angiotensin-Converting Enzyme. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Kroger, W AB - Angiotensin-Converting Enzyme (ACE) plays an essential role in blood pressure regulationand ACE inhibitors are widely used to treat cardiovascular disease. Two isoforms exist,somatic ACE (sACE) consisting of two homologous domains, N- and C-domain, and testisACE (tACE), corresponding to the C-domain of sACE. Despite a high degree of sequenceidentity, these two domains display marked differences in substrate and inhibitor specificity.Furthermore, the C-domain of ACE has been implicated to play a dominant role in bloodpressure control. It has therefore been suggested that development of ACE inhibitortreatments that selectively block the C-domain will result in decreased side-effects comparedto current therapies. Analysis of three-dimensional structures of tACE in complex withdomain-specific inhibitors has enabled the identification of key active-site residues potentiallyplaying a role in domain selectivity. To investigate the contribution of such residues, a seriesof C-domain mutants was generated containing single and multiple N-domain active-sitesubstitutions. These constructs were used to characterise specific interactions using domainselectiveinhibitors and fluorogenic peptides. Mutants tested with the fluorogenic peptidesdisplayed minimal, if any, acquisition of N-domain-like catalytic properties. Of the singlemutations, S2 (F391Y, tACE numbering) and S1 (V518T) pocket substitutions caused thelargest decreases in affinity for the C-selective phosphinic inhibitor RXPA380 (34-fold) andketo-ACE derivatives (14-26 fold), respectively. The V379S mutation caused an unexpectedincrease in affinity (2-10 fold) for C-selective inhibitors containing a P2’ Trp that could beexplained by the formation of a water-mediated hydrogen bond interaction resulting fromrearrangement of inhibitor and protein side-chains within the S2’ pocket. Multiple mutantscontaining an N-domain-like S2’ pocket combined with the S2 F391Y substitution (S2’F)caused the most notable shift in Ki from that of tACE for the highly selective phosphinicinhibitors, RXPA380 (Ki’s tACE = 69 nM; S2’F = 5300 nM) and N-specific RXP407 (Ki’stACE = 2800 nM; S2’F Ki = 16.1 nM). This work identifies key residues contributing to thedomain selectivity of ACE, and highlights the complex combination of effects involved in thisphenomenon. Furthermore, it provides useful insight for the further design of domainselectiveinhibitors. DA - 2009 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2009 T1 - A Molecular Basis for the C-Domain Selectivity of Angiotensin-Converting Enzyme TI - A Molecular Basis for the C-Domain Selectivity of Angiotensin-Converting Enzyme UR - http://hdl.handle.net/11427/3135 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/3135
dc.identifier.vancouvercitationKroger W. A Molecular Basis for the C-Domain Selectivity of Angiotensin-Converting Enzyme. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry, 2009 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/3135en_ZA
dc.language.isoeng
dc.publisher.departmentDivision of Medical Biochemistryen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.titleA Molecular Basis for the C-Domain Selectivity of Angiotensin-Converting Enzymeen_ZA
dc.typeDoctoral Thesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnamePhDen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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