Publication: Detailed functional analysis of two clinical glucose-6-phosphate dehydrogenase (G6PD) variants, G6PD<inf>Viangchan</inf>and G6PD<inf>Viangchan + Mahidol</inf>: Decreased stability and catalytic efficiency contribute to the clinical phenotype
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Issued Date
2016-06-01
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ISSN
10967206
10967192
10967192
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2-s2.0-84961990524
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Mahidol University
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SCOPUS
Bibliographic Citation
Molecular Genetics and Metabolism. Vol.118, No.2 (2016), 84-91
Suggested Citation
Usa Boonyuen, Kamonwan Chamchoy, Thitiluck Swangsri, Naowarat Saralamba, Nicholas P.J. Day, Mallika Imwong Detailed functional analysis of two clinical glucose-6-phosphate dehydrogenase (G6PD) variants, G6PD<inf>Viangchan</inf>and G6PD<inf>Viangchan + Mahidol</inf>: Decreased stability and catalytic efficiency contribute to the clinical phenotype. Molecular Genetics and Metabolism. Vol.118, No.2 (2016), 84-91. doi:10.1016/j.ymgme.2016.03.008 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/43022
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Title
Detailed functional analysis of two clinical glucose-6-phosphate dehydrogenase (G6PD) variants, G6PD<inf>Viangchan</inf>and G6PD<inf>Viangchan + Mahidol</inf>: Decreased stability and catalytic efficiency contribute to the clinical phenotype
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Abstract
© 2016 The Authors. 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, G6PDViangchanand 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 KMfor G6P of the two variants was comparable to the KMof the native enzyme, whereas the KMfor NADP+was increased 5-fold for G6PDViangchanand 8-fold for G6PDViangchan + Mahidolwhen compared with the native enzyme. Additionally, kcatof the mutant enzymes was markedly reduced, resulting in a 10- and 18-fold reduction in catalytic efficiency for NADP+catalysis for G6PDViangchanand 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 G6PDViangchanand G6PDViangchan + Mahidoland, as a consequence, contribute to the clinical phenotypes of these two clinical variants.