This study investigates the effect of the debt counselling procedure, as well as the other provisions of the National Credit Act, on the choice of the debtor or creditor to make use of the sequestration procedure in terms of the Insolvency Act. The Insolvency Act and the procedures it makes available are discussed in detail. There are two distinguishable options, namely voluntary surrender and compulsory sequestration, and particular focus is placed on the 'advantage to creditors' requirement. This requirement is of the utmost importance because the court will seldom if ever grant a sequestration order if it cannot be proven that the creditors will gain at least some form of benefit from it. The ‘advantage’ requirement is also applicable with regards to compulsory sequestration, but the onus of proof is not as strict as with voluntary surrender. The aim of this requirement is to protect creditors so that they could at least recover part of their debt. The second chapter deals with the procedure in accordance with section 74 of the Magistrates Court Act, referred to as an administration order. This is another type of debt relief available to debtors. However, there are strict requirements that have to be met before a debtor will be allowed to make use of this procedure. These requirements include that the debt owed by the debtor should not exceed R50 000 and the composition of the debt should not be complicated. This procedure is not applied very often as very few debtors owe less that R50 000. The procedures brought into life by the Credit Act are of the utmost importance for this study. The Credit Act added various new terms to the law, including overindebtedness, reckless credit, debt counselling and debt review. Each of these terms’ meaning and their effect on current procedures, as prescribed by legislation, are analysed. Following various court judgments it has become clear that the Credit Act has in fact changed the legal position drastically. In my opinion the debtor is afforded much more protection in terms of the Credit Act when it is compared to the protection that he received in terms of the Insolvency Act. Various mandatory steps were added which a creditor, or credit provider in terms of the Credit Act, must comply with before he will be allowed to claim a debt from a debtor, or consumer in terms of the Credit Act. This creates a situation where the creditor is sometimes disadvantaged since the debtor is granted a reprieve and the period he is allowed to repay his debts is often extended without the consent or input from the creditor. As soon as a debt restructuring order has been made by court, the creditor cannot take the decision on review. Regarding the relationship between insolvency procedures and debt review, it has happened on more than one occasion that creditors as well as debtors have made use of sequestration procedures only then to be forced by the courts to rather make use of debt review. This means that both creditors and debtors no longer have a choice regarding which procedure, and in accordance with which act, they would like to pursue. I am of the opinion that despite this there still is a place for both procedures in South African law. The best procedure to follow would depend on the special circumstances of each individual case. / Thesis (LL.M.)--North-West University, Potchefstroom Campus, 2012.

This study investigates the effect of the debt counselling procedure, as well as the other provisions of the National Credit Act, on the choice of the debtor or creditor to make use of the sequestration procedure in terms of the Insolvency Act. The Insolvency Act and the procedures it makes available are discussed in detail. There are two distinguishable options, namely voluntary surrender and compulsory sequestration, and particular focus is placed on the 'advantage to creditors' requirement. This requirement is of the utmost importance because the court will seldom if ever grant a sequestration order if it cannot be proven that the creditors will gain at least some form of benefit from it. The ‘advantage’ requirement is also applicable with regards to compulsory sequestration, but the onus of proof is not as strict as with voluntary surrender. The aim of this requirement is to protect creditors so that they could at least recover part of their debt. The second chapter deals with the procedure in accordance with section 74 of the Magistrates Court Act, referred to as an administration order. This is another type of debt relief available to debtors. However, there are strict requirements that have to be met before a debtor will be allowed to make use of this procedure. These requirements include that the debt owed by the debtor should not exceed R50 000 and the composition of the debt should not be complicated. This procedure is not applied very often as very few debtors owe less that R50 000. The procedures brought into life by the Credit Act are of the utmost importance for this study. The Credit Act added various new terms to the law, including overindebtedness, reckless credit, debt counselling and debt review. Each of these terms’ meaning and their effect on current procedures, as prescribed by legislation, are analysed. Following various court judgments it has become clear that the Credit Act has in fact changed the legal position drastically. In my opinion the debtor is afforded much more protection in terms of the Credit Act when it is compared to the protection that he received in terms of the Insolvency Act. Various mandatory steps were added which a creditor, or credit provider in terms of the Credit Act, must comply with before he will be allowed to claim a debt from a debtor, or consumer in terms of the Credit Act. This creates a situation where the creditor is sometimes disadvantaged since the debtor is granted a reprieve and the period he is allowed to repay his debts is often extended without the consent or input from the creditor. As soon as a debt restructuring order has been made by court, the creditor cannot take the decision on review. Regarding the relationship between insolvency procedures and debt review, it has happened on more than one occasion that creditors as well as debtors have made use of sequestration procedures only then to be forced by the courts to rather make use of debt review. This means that both creditors and debtors no longer have a choice regarding which procedure, and in accordance with which act, they would like to pursue. I am of the opinion that despite this there still is a place for both procedures in South African law. The best procedure to follow would depend on the special circumstances of each individual case. / Thesis (LL.M.)--North-West University, Potchefstroom Campus, 2012.

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

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