Rat liver cytoplasmic glucose-6-phosphate dehydrogenase. Steady-state kinetic properties and circular dichroism.
Steady-state kinetic studies including initial velocity, NADPH product inhibition, dead-end inhibition, and combined dead-end and product inhibition measurements with purified rat liver glucose-6-phosphate dehydrogenase indicate a sequential and obligatory addition of substrates in the order of NADP+, glucose-6-P for the catalytic pathway at pH 8.0. Although instability of 6-phosphoglucono-delta-lactone precluded product inhibition experiments which might directly exclude an enzyme-6-phosphoglucono-delta-lactone complex, the absence of an enzyme-glucose-6-P complex suggests that the enzyme-lactone product is unlikely and the release of products is also ordered, with NADPH released last. Consideration of the kinetic constants (Ka = 2.0 muM, Kiq = 13 muM) and cellular concentration of the substrates and products suggests extensive inhibition of the enzyme in vivo and control by the NADPH/NADP+ ratios. Circular dichroism spectra of the enzyme in 20 mM phosphate buffer at pH 7.0 and 25 degrees C indicate 51% helix and 33% pleated sheet structures which is considerably different from results (14% helix) with yeast enzymes.
D000818:Animals; D002942:Circular Dichroism; D003593:Cytoplasm / Q000201:enzymology*; D005954:Glucosephosphate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008433:Mathematics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011487:Protein Conformation; D051381:Rats
D005954:Glucosephosphate Dehydrogenase