Expression and biochemical characterization of human glucose-6-phosphate dehydrogenase in Escherichia coli: a system to analyze normal and mutant enzymes.
We have developed a system to characterize normal and mutated glucose-6-phosphate dehydrogenase (G6PD) enzymes in vitro. Normal or mutant G6PD cDNA was subcloned into a pGEX-3X vector, which allowed production of a functional fusion protein in Escherichia coli. When we compared the recombinant normal enzyme with authentic human G6PD, indistinguishable Km values for glucose-6-phosphate (G6P) and NADP were obtained, and the utilization rates for two substrate analogues (2-deoxy G6P and deamino NADP) also showed no difference between the enzymes. This system was used to assay a biochemically uncharacterized variant, G6PD Taipei (493 A-->G; 165 Asn-->Asp), plus two other known mutations (487 G-->A; 163 Gly-->Ser and 592 C-->T; 198 Arg-->Cys) that are located close to or within the putative G6P binding domain. Our results show that the G6PD activities of these three mutants were greatly reduced. No significant alteration in G6PD kinetics was observed for both 487 and 493 mutations. However, a drastic reduction in the Km for G6P (4-fold decrease) and tremendous increases in utilization rates of 2-deoxy G6P (32-fold increase) and deamino NADP (6-fold increase) were associated with the 592 mutation. This results suggests that arginine 198 in human G6PD, possibly located within the putative G6P binding domain, may play an important role in binding the substrate G6P. In addition, we and others have recently identified that at least nine different types of mutations are responsible for G6PD deficiency in Chinese. In this report, we also present the occurrence rate of each mutation present in the population of Taiwan.
Tang
T K
TK
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China.
Yeh
C H
CH
Huang
C S
CS
Huang
M J
MJ
D001483:Base Sequence; D004926:Escherichia coli; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013624:Taiwan
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005954:Glucosephosphate Dehydrogenase