Investigation of the bi-substrate kinetics of a recombinant human glycine N-acyltransferase with a known non-synonymous single nucleotide polymorphism (N156S)
Biotransformation is fast becoming a buzzword of our time. This is due to the ever increasing exposure of humans to xenobiotic substances. The detoxification of these xenobiotics occur via various detoxifying mechanisms in which these substances are converted to more hydrophilic forms, to ease their excretion from the body, and thus also to avoid the possibly toxic accumulation of these compounds in the body. One such detoxifying mechanism is the glycine conjugation pathway. The focus of this study was on one of the enzymes involved in this pathway responsible for the detoxification of metabolites by conjugation to glycine, the glycine N-acyltransferase (GLYAT) enzyme. Interindividual variation has been observed in the glycine conjugation pathway, which may involve various contributing factors. One such factor is the genetic variation in the GLYAT gene. Single nucleotide polymorphisms (SNPs) occurring in the exon regions of the GLYAT gene have been associated with altered enzyme activities. This study focused on the N156S human GLYAT. In previous studies, this variant was found to have the highest enzyme activity as well as the highest allele frequency, and is now regarded as the wild-type variant of human GLYAT. Thus, making this variant exceptionally relevant for investigations. In this study a bacterially codon optimised N156S human GLYAT construct was generated by means of site-directed mutagenesis and the protein expressed with an N-terminal histidine tag for affinity purification. Throughout this process, some optimisation experiments were also conducted. These included the optimisation of the protein expression, extraction and storage conditions. The enzyme kinetic studies were then conducted with the purified recombinant N156S human GLYAT in the presence of two varying substrates (benzoylcoenzyme A and glycine) to be able to characterise the bi-substrate kinetic parameters of this human GLYAT variant. The Km value were 49± 13 μM for benzoyl-coenzyme A and 20 ± 4 mM for glycine. These findings correlated with the values found in the literature which reported Km values for benzoyl-coenzyme A ranging from 6 to 67 μM; and from 6.4 to 26.6 mM for glycine. The kinetic model we proposed, the random order sequential mechanism, which is a ternary complex mechanism, also agreed to what had previously been described for GLYAT enzymes. The N156S human GLYAT enzyme activity was further characterised by quantifying the amount of product, hippuric acid (HA) formed in the presence of varying substrate concentrations. This was done using an HPLC-MS/MS method with a stable isotope as internal standard. The findings suggested that increasing amounts of HA was formed when the glycine substrate concentrations were increased, and benzoyl-coenzyme A concentrations were kept constant. This effect, however, was only noticed up to a certain glycine concentration, after which the HA formation slowed down and reached a plateau. These findings suggested that the GLYAT enzyme had evolved with a limited rate of detoxification, possibly to avoid glycine depletion. Glycine is used for the production of creatine, bile salts, porphyrins, collagen, elastin, glutathione, as well as other proteins, and the depletion of glycine stores may have serious implications. Further characterisation of this and other variants of GLYAT is however necessary, in order to fully understand the catalytic mechanisms of this enzyme. Once enough knowledge of these variants and their enzymatic properties are known, it may then become possible to attempt to design a GLYAT with altered substrate specificity, which may be useful for the treatment of organic acidemias.