Title
Detailed functional analysis of two clinical glucose-6-phosphate dehydrogenase (G6PD) variants, G6PDViangchan and G6PDViangchan + Mahidol: Decreased stability and catalytic efficiency contribute to the clinical phenotype
Author
Usa Boonyuen, Kamonwan Chamchoy, Thitiluck Swangsri, Naowarat Saralamba, Nicholas P.J. Day, Mallika Imwong
Year
2016
Journal
Molecular Genetics and Metabolism
Abstract
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, G6PDViangchan and G6PDViangchan + 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 KM for G6P of the two variants was comparable to the KM of the native enzyme, whereas the KM for NADP+ was increased 5-fold for G6PDViangchan and 8-fold for G6PDViangchan + Mahidol when compared with the native enzyme. Additionally, kcat of the mutant enzymes was markedly reduced, resulting in a 10- and 18-fold reduction in catalytic efficiency for NADP+ catalysis for G6PDViangchan and G6PDViangchan + 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 G6PDViangchan and G6PDViangchan + Mahidol and, as a consequence, contribute to the clinical phenotypes of these two clinical variants.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Protein denaturation, Thermodynamics, Biochemistry, Medicinal