Dog liver glucose-6-phosphate dehydrogenase:: purification and kinetic properties

Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in liver. G6PD was purified from dog liver with a specific activity of 130 U mg(-1) and a yield of 18%. PAGE showed two bands on protein staining; only the Slower moving band had G6PD activity. The observation of one band on SDS/PAGE with M-r of 52.5 kDa suggested the faster moving band on native protein staining was (lie monomeric form of the enzyme. Dog liver G6PD had a pH optimum of 7.8. The activation energy, activation enthalpy, and Q(10), for the enzymatic reaction were calculated to be 8.96, 8.34 kcal mol(-1), and 1,62, respectively. The enzyme obeyed "Rapid Equilibrium Random Bi Bi" kinetic model with K-m values of 122 +/- 18 muM for glucose-6-phosphate (G6P) and 10 +/- 1 muM for NADP. G6P and 2-deoxyglucose-6-phosphate were used with catalytic efficiencies (k(cat)/K-m) of 1.86 x 10(6) and 5.55 x 10(6) M-1 s(-1). respectively. The intrinsic K-m value for 2-deoxyglucose-6- phosphate was 24 +/- 4 mM. Deamino-NADP (d-NADP) could replace NADP as coenzyme. With G6P as cosubstrate, K-m d-ANADP was 23 +/- 3 mM; K-m for G6P remained the same Lis with NADP as coenzyme (122 +/- 18 muM). The catalytic efficiencies of NADP and d-ANADP (G6P as substrate) were 2.28 x 10(7) and 6.76 x 10(6) M-1 s(-1), respectively. Dog liver G6PD,,vas inhibited competitively by NADPH (K-i = 12.0 +/- 7.0 muM). Low K-i indicates tight enzyme:NADPH binding and the importance of NADPH in the regulation of the pentose phosphate pathway. (C) 2002 Published by Elsevier Science Ltd.