Detailed functional analysis of two clinical glucose-6-phosphate dehydrogenase (G6PD) variants, G6PD(Viangchan) and G6PD(Viangchan) (+) (Mahidol): Decreased stability and catalytic efficiency contribute to the clinical phenotype

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is an X-linked hereditary genetic defect that is the most common polymorphism and enzymopathy in humans. To investigate functional properties of two clinical variants, G6PD(Viangchan) and G6PD(Viangchan) (+) (Mahidol), these two mutants were created by overlap-extension PCR, expressed in Escherichia coli and purified to homogeneity. We describe an overexpression and purification method to obtain substantial amounts of functionally active protein. The K(M) for G6P of the two variants was comparable to the K(M) of the native enzyme, whereas the K(M) for NADP(+) was increased 5-fold for G6PD(Viangchan) and 8-fold for G6PD(Viangchan) (+) (Mahidol) when compared with the native enzyme. Additionally, k(cat) of the mutant enzymes was markedly reduced, resulting in a 10- and 18-fold reduction in catalytic efficiency for NADP(+) catalysis for G6PD(Viangchan) and G6PD(Viangchan) (+) (Mahidol), respectively. Furthermore, the two variants demonstrated significant reduction in thermostability, but similar susceptibility to trypsin digestion, when compared with the wild-type enzyme. The presence of NADP(+) is shown to improve the stability of G6PD enzymes. This is the first report indicating that protein instability and reduced catalytic efficiency are responsible for the reduced catalytic activity of G6PD(Viangchan) and G6PD(Viangchan) (+) (Mahidol) and, as a consequence, contribute to the clinical phenotypes of these two clinical variants.