Purification and kinetic characterization of 6-phosphogluconate dehydrogenase from Schizosaccharomyces pombe.
6-Phosphogluconate dehydrogenase is the pivotal enzyme that links the gluconate route and the oxidative phase of the pentose phosphate pathway in Schizosaccharomyces pombe. The enzyme differs from the known 6-phosphogluconate dehydrogenases of other sources in that the Schizosaccharomyces enzyme is tetrameric having a subunit mass of 38 kDa, that it requires NADP+ obligatorily for activity, and that it can be activated by divalent metal ions such as Co2+ and Mn2+. Steady-state kinetic studies were undertaken. Initial rate and product inhibition results suggest that 6-phosphogluconate dehydrogenase from Schizosaccharomyces pombe catalyzes NADP(+)-linked oxidative decarboxylation of 6-phosphogluconate by an equilibrium random mechanism with two independent binding sites, namely one site for the nicotinamide coenzyme, NADP+/NADPH, and another site for 6-phosphogluconate-D-ribulose-5-phosphate and for CO2. Studies of pH dependence implicated a basic residue with a pK value of 7.4 in the binding of 6-phosphogluconate and an acidic residue with a pK value of 6.7 in the cation-mediated interaction of NADP+ with the enzyme.
Tsai
C S
CS
Department of Chemistry, Carleton University, Ottawa, ON, Canada. stsai@ccs.carleton.ca
Chen
Q
Q
D003653:Decarboxylation; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015233:Models, Statistical; D009249:NADP / Q000378:metabolism; D010734:Phosphogluconate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification*; D012568:Schizosaccharomyces / Q000201:enzymology*
D009249:NADP; D010734:Phosphogluconate Dehydrogenase