Combined effects of double mutations on catalytic activity and structural stability contribute to clinical manifestations of glucose-6-phosphate dehydrogenase deficiency

Phonchanan Pakparnich,Kamonwan Chamchoy,Sirapapha Sudsumrit,Ubolsree Leartsakulpanich,Teeraporn Suteewong,Usa Boonyuen,Mallika Imwong,Danaya Pakotiprapha

doi:10.1038/s41598-021-03800-z

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans, affecting ~ 500 million worldwide. A detailed study of the structural stability and catalytic activity of G6PD variants is required to understand how different mutations cause varying degrees of enzyme deficiency, reflecting the response of G6PD variants to oxidative stress. Furthermore, for G6PD double variants, investigating how two mutations jointly cause severe enzyme deficiency is important. Here, we characterized the functional and structural properties of nine G6PD variants: G6PD Gaohe, G6PD Mahidol, G6PD Shoklo, G6PD Canton, G6PD Kaiping, G6PD Gaohe + Kaiping, G6PD Mahidol + Canton, G6PD Mahidol + Kaiping and G6PD Canton + Kaiping. All variants were less catalytically active and structurally stable than the wild type enzyme, with G6PD double mutations having a greater impact than single mutations. G6PD Shoklo and G6PD Canton + Kaiping were the least catalytically active single and double variants, respectively. The combined effects of two mutations were observed, with the Canton mutation reducing structural stability and the Kaiping mutation increasing it in the double mutations. Severe enzyme deficiency in the double mutants was mainly determined by the trade-off between protein stability and catalytic activity. Additionally, it was demonstrated that AG1, a G6PD activator, only marginally increased G6PD enzymatic activity and stability.

Highlights

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans, affecting ~ 500 million worldwide
The kcat values of Class II variants [G6PD Gaohe (His32Arg), G6PD Canton (Arg459Leu) and G6PD Kaiping (Arg463His)] were reduced by approximately 50% and those of the double mutations were lower than the single mutations with G6PD Gaohe + Kaiping (His32Arg + Arg463His) and G6PD Canton + Kaiping (Arg459Leu + Arg463His) having a 15-fold decrease when compared with the kcat of G6PD wild type (WT)
Other variants showed similar binding affinity toward the G6P substrate when compared with the binding affinity of the WT enzyme, G6PD Canton, G6PD Mahidol + Canton (Gly163Ser + Arg459Leu) and G6PD Canton + Kaiping had lower Km values (1.5–2.6-fold)

Summary

Introduction

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans, affecting ~ 500 million worldwide. A detailed study of the structural stability and catalytic activity of G6PD variants is required to understand how different mutations cause varying degrees of enzyme deficiency, reflecting the response of G6PD variants to oxidative stress. For G6PD double variants, investigating how two mutations jointly cause severe enzyme deficiency is important. All variants were less catalytically active and structurally stable than the wild type enzyme, with G6PD double mutations having a greater impact than single mutations. G6PD Shoklo and G6PD Canton + Kaiping were the least catalytically active single and double variants, respectively. G6PD deficiency can lead to a decrease in NADPH and reduced glutathione levels, causing oxidative d amage[1] Under this condition, red blood cells become susceptible to hemolysis. We examined the effect of AG1 on enzyme activity and protein stability of the G6PD variants

Methods

Results

Discussion

Conclusion