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TK Tang, CH Yeh, CS Huang and MJ Huang
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic
of China.
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.
This article has been cited by other articles:
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| Copyright © 1994 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
