33008-07-0Relevant articles and documents
Enzymatic characterization and elucidation of the catalytic mechanism of a recombinant bovine glycine N-acyltransferase
Badenhorst, Christoffel P. S.,Jooste, Maritza,Van Dijk, Alberdina A.
experimental part, p. 346 - 352 (2012/06/30)
Glycine conjugation, a phase II detoxification process, is catalyzed by glycine N-acyltransferase (GLYAT; E.C. 2.3.1.13). 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 Glu 226 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. Copyright
N-Acylglycines: Gas chromatographic mass spectrometric identification and determination in urine by selected ion monitoring
Gregersen,Keiding,Kolvraa
, p. 439 - 443 (2007/10/05)
Eleven biologically interesting N-acylglycines have been synthesized and the gas chromatographic and mass spectrometric properties of their trimethylsilyl derivatives studied. A sharp and reproducible gas chromatographic peak could be obtained for each n-acylglycine as the N,O-bis(trimethylsilyl)-N-acylglycine. By the use of these derivatives a sensitive and specific selected ion monitoring method for the determination of N-acylglycines in human urine has been developed.
