Human hereditary glutathione synthetase deficiency: kinetic properties of mutant enzymes

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Abstract
Patients with hereditary glutathione synthetase deficiency suffer from haemolytic anaemia, 5-oxoprolinuria, metabolic acidosis, recurrent bacterial infections and various degrees of central nervous system dysfunction. To investigate the molecular basis of the mutations associated with this disease, seven naturally occurring missense mutations [L188P (Leu188→Pro), D219A, D219G, Y270C, Y270H, R283C and P314L] were expressed using a His-tagged, Escherichia coli-based expression system. Effects of the mutations on kinetic properties, including negative co-operative binding of γ-glutamyl substrate, were evaluated. The mutation P314L did not have any major effect on these parameters and was classified as a neutral mutation. The remaining mutations decreased Vmax to 2–27% of wild-type activity. Negative co-operativity for γ-gluABA (L-γ-glutamyl-L-α-aminobutyric acid) was abolished in five mutant recombinant enzymes, whereas for one mutant enzyme, this co-operativity changed from negative to positive. The structural consequences of the mutations were interpreted on the basis of the known structure of the wild-type enzyme.