Biochemical Analysis of Two Single Mutants that Give Rise to a Polymorphic G6PD A-Double Mutant

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme that plays a crucial role in the regulation of cellular energy and redox balance. Mutations in the gene encoding G6PD cause the most common enzymopathy that drives hereditary nonspherocytic hemolytic anemia. To gain insights into the effects of mutations in G6PD enzyme efficiency, we have investigated the biochemical, kinetic, and structural changes of three clinical G6PD variants, the single mutations G6PD A+ (Asn126AspD) and G6PD Nefza (Leu323Pro), and the double mutant G6PD A− (Asn126Asp + Leu323Pro). The mutants showed lower residual activity (≤50% of WT G6PD) and displayed important kinetic changes. Although all Class III mutants were located in different regions of the three-dimensional structure of the enzyme and were not close to the active site, these mutants had a deleterious effect over catalytic activity and structural stability. The results indicated that the G6PD Nefza mutation was mainly responsible for the functional and structural alterations observed in the double mutant G6PD A−. Moreover, our study suggests that the G6PD Nefza and G6PD A− mutations affect enzyme functions in a similar fashion to those reported for Class I mutations.

Effects of Single and Double Mutants in Human Glucose-6-Phosphate Dehydrogenase Variants Present in the Mexican Population: Biochemical and Structural Analysis

“…it increased 1.2-, 1.3-, and 1.8-fold, respectively, with respect to WT G6PD. These results are in concordance with fluorescence intensity obtained previously for Class I mutants G6PD Zacatecas and Durham, Class II G6PD Nefza, and double mutant G6PD A, respectively, where fluorescence intensity was increased two-fold [11,15]. As previously noted, increased fluorescence intensity could suggest modifications in the native 3D structure, which has a negative effect on the catalytic activity in the four clinical mutants.…”

“…The kinetic parameters of the recombinant enzymes G6PD A+ (Asn126Asp), G6PD San Luis Potosi (Asn126Tyr), G6PD Guadalajara (Arg387Cys), and G6PD Mount Sinai (Asn126Tyr/Arg387Cys) were determined spectrophotometrically at 25 • C when monitoring the reduction of the NADP+ substrate into an absorbance of 340 nm [11,15,19,25,33]. Standard activity assay was performed in a final volume of 1 mL of the reaction in buffer T (Tris-HCl 0.1 mM, pH 8.0, and MgCl 2 3 mM).…”

“…This mutation results in a change of adenine by guanine (A→G) in nucleotide (nt) 376 of the G6PD gene and is located in exon 5, with a change on the asparagine by aspartic acid (Asn→Asp) amino acid residue in the 126 position of the three-dimensional (3D) structure (Figure 1). It is interesting to note that the mutation is far from both the active site and the interface region of the protein; nevertheless, the asparagine 126 in the wild-type form of the enzyme is part of the α-helix structure exposed in an aqueous medium [15,16] Int. J. Mol.…”

“…2020, 21, x FOR PEER REVIEW 3 of 25 a change of adenine by guanine (AG) in nucleotide (nt) 376 of the G6PD gene and is located in exon 5, with a change on the asparagine by aspartic acid (AsnAsp) amino acid residue in the 126 position of the three-dimensional (3D) structure (Figure 1). It is interesting to note that the mutation is far from both the active site and the interface region of the protein; nevertheless, the asparagine 126 in the wild-type form of the enzyme is part of the α-helix structure exposed in an aqueous medium [15,16] Class II G6PD San Luis Potosi was reported for the first time in a study which included 5000 Mexican individuals, where the G6PD deficiency was determined in the Mexican population. This study was carried out across the country and the variant was detected in an anonymous blood sample from San Luis Potosi State.…”

“…This mutant showed a single nucleotide substitution of adenine for thymine (A→T) at nt 376 (exon 5), which results in the substitution of asparagine by tyrosine 126 (Asn→Tyr) amino acid residue [17] (Figure 1). This variant showed that the mutation is localized in the same position (nt 376) as the G6PD A+ variant [15]. The single Class I G6PD Guadalajara variant shows the substitution of cytosine by thymine (C→T) at nt 1159 (exon 10), which gives a change in the arginine residue by cysteine 387 (Arg→Cys) located in the binding site of structural NADP+ of the 3D structure, which is implicated in enzyme stability and dimer formation.…”