Characterization of the enzyme activity and substrate specificity of GLYAT haplotypes

MSc (Biochemistry), North-West University, Potchefstroom Campus

The unavoidable exposure to xenobiotics makes detoxification a necessity. Detoxification is not just one reaction but four reactions (Phase 0, I, II and III) that work together. The glycine conjugation pathway is one of the Phase II detoxification pathways. The glycine conjugation pathway plays an important role in detoxification xenobiotic substances such as benzoate and salicylate. Benzoate can be found in foods and drinks in the form of additives and preservatives and salicylate is found in aspirin. This pathway is responsible for making the substances less lipophilic for ease of excretion in the urine. The glycine conjugation pathway is made up of two different enzymes, the medium chain fatty acid: CoA ligase (ASCM2B, E.C. 6.2.1.2) enzyme and the glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13) enzyme. This pathway is theorised to be critical for survival as there are only 14 haplotypes, indicating that the pathway is highly conserved, and no defects in this pathway have been reported. This study focused on the second enzyme GLYAT, which is responsible for the conjugation of substances with glycine. GLYAT has the highest substrate affinity for benzoyl-CoA compared to other substances such as salicyl-CoA, isovaleryl-CoA, propionyl-CoA and butyryl-CoA. Previous studies found that single nucleotide polymorphisms (SNPs) can effect enzyme activity. The S156 variant is the suggested wild-type variant with the highest relative enzyme activity and the highest allele frequency. The S17T variant had the third highest relative enzyme activity and the R199C variant was barely active. The aim of this study was to evaluate whether the conserved haplotypes (S156, T17S156 and S156C199), that were identified in the worldwide genetic variation analyses, have similar effects on the enzyme activity as the SNPs that were characterised. The kinetic mechanism of GLYAT in previous studies used one substrate at saturating concentration while varying the other substrate. In this study both substrates, benzoyl-CoA and glycine, were varied using a wide range of concentrations. This S156 and S156C199 variants were already available in the laboratory. The T17S156 variant was constructed using site directed mutagenesis with the S156 variant template. All three proteins were successfully expressed in Origami_pGro7 cells and purified using N-terminal Trx-His-tags. The purification was needed as purified proteins are needed for enzyme kinetic reactions. Relative enzyme activity for all three proteins were determined, S156 had the highest relative enzyme activity when compared to the other two variants (4.19μM/min). T17S156 had the second highest activity (2.6μM/min) followed by S156C199 with the lowest activity (1.7μM/min). The bi-substrate kinetics was determined with varying substrate concentrations after a global fit of data was performed, the sigmoidal enzyme kinetic model had the best fit. The substrate specificity was calculated and S156 had the highest substrate specificity for glycine of 19.89 s-1mM-1 and for benzoyl-CoA of 4.46 s4 1mM-1, T17S156 had the second highest substrate specificity for both glycine and benzoyl-CoA with 11.01 s-1mM-1 and 2.68 s-1mM-1 respectively. S156C199 had the lowest substrate specificity values with 1.65 s-1mM-1 for glycine and 0.66 s-1mM-1 for benzoyl-CoA. The kinetic studies can help to classify GLYAT with regards to how the different haplotypes will have an influence on the enzyme activity as well as the substrate specificity in order to help understand the function of the enzyme in vivo.

Masters