Despite being the first biochemical reaction to be discovered, the glycine conjugation pathway remains poorly
characterised. It has generally been assumed that glycine conjugation serves to increase the water solubility of
organic acids, such as benzoic acid and isovaleric acid, in order to facilitate urinary excretion of these compounds.
However, it was recently suggested that the conjugation of glycine to benzoate should be viewed as a
neuroregulatory process that prevents the accumulation of glycine, a neurotransmitter, to toxic levels. The true
importance of glycine conjugation in metabolism is therefore not well understood. However, no genetic defect of
glycine conjugation has ever been reported. This seems to suggest that glycine conjugation is a fundamentally
important metabolic process, whatever its function may be. Therefore, a major objective of this thesis was to
develop a deeper understanding of glycine conjugation and its metabolic significance. A review of the literature on
GLYAT and glycine conjugation suggested that the primary purpose of glycine conjugation is indeed to detoxify
benzoate and other aromatic acids of dietary origin. However, the commonly held assumption, that glycine
conjugation increases the water solubility of aromatic acids in order to facilitate urinary excretion, seems to be
incorrect. A better explanation for the detoxification of benzoate by means of glycine conjugation is based on
hydrophilicity, not water solubility. Because of its lipophilic nature, benzoic acid is capable of passively diffusing
across the mitochondrial inner membrane into the matrix space, where it accumulates due to the pH gradient
over the inner membrane. Although benzoate can be exported from the matrix by organic anion transporters, this
process would likely be futile because benzoic acid can simply diffuse back into the matrix. Hippurate, however, is
significantly less lipophilic and therefore less capable of diffusing into the matrix. It is therefore not transport out
of the mitochondrial matrix that is facilitated by glycine conjugation, but rather the ability of the glycine
conjugates to re-enter the matrix that is decreased.
The conversion of benzoate to hippurate is a two-step process. First, benzoate is activated by an ATP-dependent
acid:CoA ligase (ACSM2A) to form the more reactive benzoyl-CoA. Second, glycine N-acyltransferase (GLYAT)
catalyses the formation of hippurate and CoASH from benzoyl-CoA and glycine. Another major objective of this thesis was to gain a better understanding of the structure and function of the GLYAT enzyme. While the substrate
selectivity and enzyme kinetics of GLYAT have been investigated to some extent, almost nothing has been
published on the structure, active site, or catalytic mechanism of GLYAT. Furthermore, while interindividual
variation in the rate of glycine conjugation has been reported by several researchers, it is not known if, or how,
genetic variation in the human GLYAT gene contributes to this interindividual variation. To address these issues,
systems for the bacterial expression of recombinant bovine GLYAT and recombinant human GLYAT were
developed. Because no crystal structure of GLYAT has been reported, homology modelling was used to generate a
molecular model of bovine GLYAT. By comparing the molecular model to other acyltransferases for which the
catalytic residues were known, Glu227 of bovine GLYAT was identified as a potential catalytic residue. Site directed
mutagenesis was used to generate an E227Q mutant recombinant bovine GLYAT lacking the proposed catalytic
residue. Characterisation of this mutant suggested that Glu227 was indeed the catalytic residue, and the GLYAT
catalytic mechanism was elucidated. The molecular model was also used to identify Asn131 of bovine GLYAT as a
potential active site residue. Site-directed mutagenesis was used to generate an N131C mutant, which was
sensitive to inhibition by the sulfhydryl reagent DTNB. This suggests that the Asn131 residue of bovine GLYAT may
be situated in the active site of bovine GLYAT, but more work is needed to confirm this result. Finally, site-directed
mutagenesis was used to generate variants of recombinant human GLYAT corresponding to six of the known SNPs
in the human GLYAT gene. Expression and characterisation of the recombinant human GLYAT variants revealed
that the enzyme activity and KM (benzoyl-CoA) parameter of the recombinant human GLYAT were influenced by
SNPs in the human GLYAT gene. This suggests that genetic variation in the human GLYAT gene could partly explain
the interindividual variation in the rate of glycine conjugation observed in humans. Interestingly, the SNPs that
negatively influenced enzyme activity also had low allele frequencies, suggesting that there may be some selective
advantage to having high GLYAT activity. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014
| Identifer | oai:union.ndltd.org:NWUBOLOKA1/oai:dspace.nwu.ac.za:10394/10853 |
| Date | January 2014 |
| Creators | Badenhorst, Christoffel Petrus Stephanus |
| Source Sets | North-West University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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