BIOCHEMICAL STUDIES OF GENETIC VARIANTS OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE IN NIGERIA

Abstract

Comparative biochemical studies have been carried out on five genetic variants of human erythrocyte glucose-6-phosphate dehydrogenase in Nigeria. These variants A, A-, B, Ijebu-Ode and Ita-Bale in order of decreasing electrophoretic mobility, are found with different frequencies in Nigeria. The first three variants are common (A- is associated with deficiency) while the latter two are rare (below polymorphic frequency) and have been characterised for the first time. (2) Partially purified preparations of the five enzyme types, have been used in a number of kinetic studies. All the variants have approximately the same affinity for glucose-6-phosphate but when the substrate analogue 2-deoxy glucose-6-phosphate is used, Ijebu-Ode emerges as the variant with the lowest affinity for it. The effect of pH on the activities of the two rare variants (carried out according to WHO recommendation) indicate that both of them have a slightly biphasic pattern. The rate of inactivation of the two rare variants as compared with the B variant show that Ita-Bale had a minimally increased thermolability while the thermolability of Ijebu-Ode was markedly increased. (3) A detailed analysis of how pH affects two main kinetic parameters of enzyme reactions, namely Vmax and Km, for glucose 6-phosphate has been carried out for all the variants. For all pH (using tris-borate-citrate, tris-borate, tris-borate-buffers) over the entire range explored from pH 5.5 to 9.5. In addition, A, Ijebu-Ode and Ita-Bale have a plateau in the pH region between 7.5 and 8.2, whereas A and B have a ‘peak and trough’ in the same region. Characteristic patterns were obtained for each variant on plotting log Vmax, log Km and log Vmax / Km versus pH. From the waves in the plots, the pKs corresponding to ionised imidazolium and thiol residues in free enzymes and enzyme-substrate complexes were obtained. The data also indicate the absence of thiol residue in or near the active centre of Ijebu-Ode. The Km data obtained for type A- have not been always consistent and a possible explanation is presented. (4) The dependence of enzyme activity on NADP and NADPH concentration was examined for the five variants. Sigmoidal saturation kinetics of A, B and Ita-Bale variants for NADP clearly differentiate them from the near-hyperbolic kinetics of A and Ijebu-Ode enzyme types. Two dissociation constants (Ks₁ and Ks2 ), corresponding to states of low and high affinity for NADP, have been, obtained for each variant. An explanation about the mechanism of such interactions is also presented. The complex kinetics of interaction of the enzyme variants with NADPH has implicated this metabolite as both an activator and inhibitor, but A- type emerges as the least activated and inhibited. The sum total of the interactions of G6PD with NADP and its metabolite, NADPH, has led to the suggestion that G6PD is a regulatory (allosteric) protein. (5) The five genetic variants exhibit differential interactions with some potent inhibitors which bind to the sulphydryl groups, like N-ethylmaleimide and p-chloromercuribenzoate. Of all the variants, Ijebu-Ode is strickingly resistant to PCMB and totally resistant to NEM. An explanation for the resistance of Ijebu-Ode to these inhibitors is suggested. (6) Enzyme inactivation as a function of temperature exhibits distinctive profiles for all the variants and Ijebu-Ode particularly displayed a unique picture. The implication of the differences observed in this thermal inactivation profile studies are also discussed. (7) Finally, it is suggested that differences observed in the catalytic and kinetic properties among the five variants of G6PD are a reflection of the structural differences among them which also implies different mutational changes in the structural locus for red cell glucose-6-phosphate dehydrogenase.