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dc.contributor.authorBadenhorst, Christoffel P.S.
dc.contributor.authorJooste, Maritza
dc.contributor.authorVan Dijk, Alberdina A.
dc.identifier.citationBadenhorst, C.P.S. et al. 2012.Enzymatic characterization and elucidation of the catalytic mechanism of a recombinant Bovine Glycine N-Acyltransferase. Drug metabolism and disposition, 40(2):346-352. []en_US
dc.description.abstractGlycine conjugation, a phase II detoxification process, is catalyzed by glycine N-acyltransferase (GLYAT; E.C. GLYAT detoxifies various xenobiotics, such as benzoic acid, and endogenous organic acids, such as isovaleric acid, which makes GLYAT important in the management of organic acidemias in humans. We cloned the open reading frame encoding the bovine ortholog of GLYAT from bovine liver mRNA into the bacterial expression vector pColdIII. The recombinant enzyme was expressed, partially purified, and enzymatically characterized. Protein modeling was used to predict Glu226 of bovine GLYAT to be catalytically important. This was assessed by constructing an E226Q mutant and comparing its enzyme kinetics to that of the wild-type recombinant bovine GLYAT. The Michaelis constants for benzoyl-CoA and glycine were determined and were similar for wild-type recombinant GLYAT, E226Q recombinant GLYAT, and GLYAT present in bovine liver. At pH 8.0, the E226Q mutant GLYAT had decreased activity, which could be compensated for by increasing the reaction pH. This suggested a catalytic mechanism in which Glu226 functions to deprotonate glycine, facilitating nucleophilic attack on the acyl-CoA. The recombinant bovine GLYAT enzyme, combined with this new understanding of its active site and reaction mechanism, could be a powerful tool to investigate the functional significance of GLYAT sequence variations. Eventually, this should facilitate investigations into the impact of known and novel sequence variations in the human GLYAT gene.en_US
dc.titleEnzymatic characterization and elucidation of the catalytic mechanism of a recombinant Bovine Glycine N-Acyltransferaseen_US
dc.contributor.researchID10997938 - Van Dijk, Alberdina Aike
dc.contributor.researchID21489459 - Badenhorst, Christoffel Petrus Stephanus
dc.contributor.researchID13013718 - Jooste, Maritza

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