Supervisors: Dr Ed Sturrock and Professor Ravi Acharya
Keywords: angiotensin-converting enzyme (ACE), kinetics, structure, mutagenesis.
Aims: To determine the structure of under-glycosylated, truncated glycoforms of ACE using x-ray crystallography.
Background: Angiotensin-Converting Enzyme (ACE) is a metalloprotease that plays a pivotal role in blood pressure, electrolyte and fluid homeostasis through the conversion of angiotensin I to the active vasopressor, angiotensin II [cite num=1]. Somatic ACE (sACE) contains two homologous domains: A C-domain and an N-Domain [cite num=2]. The rational design of domain-specific ACE-inhibitors in the treatment of cardiovascular diseases is dependent upon a detailed structural knowledge of the molecule. Recently the crystal structure of a minimally glycosylated C-domain has been determined [cite num=3] and it has been shown that extensive deglycosylation does not influence the structure or activity of the enzyme[cite num=4].
Objectives and research plan: A truncated ACE protein, lacking the cytosolic tail and transmembrane domain, has been deglycosylated using an in vitro site-directed mutagenesis approach whereby selected Asn-residues were mutated to Gln [cite num=4]. Mutants will be expressed in Chinese Hamster Ovary (CHO) cells and purified using affinity chromatography methodology established in our laboratory. The Km and kcat kinetic parameters of under-glycosylated glycoforms will be determined using domain-specific substrates and compared to that of fully-glycosylated N-domain. Extensive crystallization trials will be carried out using commercially available crystal screen conditions (Hampton Research). In addition, ammonium sulfate, PEG (FLUKA), and MPD (Sigma) matrices will be used. Collection of X-ray diffraction data will be carried out on the crystals, locally at the University of the Western Cape, and at the Daresbury synchrotron source in the UK. The structure will be solved using the molecular replacement method. Model building and refinement will be carried out using the programs O and CNS28, respectively.
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- Gordon, K., Redelinghuys, P., Schwager, S. L., Ehlers, M. R., Papageorgiou, A. C., Natesh, R., . . . Sturrock, E. D. (2003). Deglycosylation, processing and crystallization of human testis angiotensin-converting enzyme. Biochem J, 371(Pt 2), 437-42. PMID: 12542396 [cite num=”4″ return=TRUE]