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The steady state kinetics of pig liver glucose-6-phosphate dehydrogenase is consistent with an ordered, sequential mechanism in which NADP is bound first and NADPH released last. Kia is 9.0 muM, Ka is 4.8 muM, and Kb is 36 muM. Glucosamine 6-phosphate, a substrate analogue and competitive inhibitor, is used to help rule out a possible random mechanism. ADP is seen to form a complex with the free form of the enzyme whereas ATP forms a complex with both the free and E-NADP forms of the enzyme. The KI for the E-ADP complex is 1.9 mM, while the Ki values for the E-ATP and E-NADP-ATP complexes are 7.2 and 4.5 mM, respectively.

A method is described for the isolation and purification of 6-phosphogluconate dehydrogenase from pig liver. The molecular weight is estimated at 83,000 and that of the subunits is 42,000 as determined by gel electrophoresis. The pH maximum is 8.5 in 50 mM glycine/NaOH buffer and from 7.5 to 10 in 50 mM phosphate buffer at 30 degrees. Magnesium ion is not required for activity and acts as an inhibitor at concentrations above 20 mM. A cellular fractionation study indicates that this enzyme is located almost entirely within the soluble portion of the cytoplasm. Kinetic studies have been done in 50 mM glycine buffer, pH 8.5, at 30 degrees. The data are consistent with a sequential mechanism in which NADP+ is added first, followed by 6-phosphogluconate, and the products are released in the order, CO2, ribulose 5-phosphate, and NADPH. The Michaelis constant is 13.5 muM for 6-phosphogluconate. Dissociation constants are 4.8 muM for NADP+ and 5.1 muM for NADPH.

A method is described for the isolation and purification of glucose dehydrogenase from bovine liver in which Triton X-100 is used to release glucose dehydrogenase from the endoplasmic reticulum. This particular form of the enzyme contains a limited amount (1.5%) of cholesterol, fatty acids, triglycerides, and cholesterol esters. The Circular Dichroism data analysis indicates 52% alpha helix, 15% beta pleated sheet, and 33% random for this enzyme. The molecular weight is estimated at 235,000 and that of the subunits is 59,000 as determined by gel electrophoresis. The pH of maximum activity is 9.5 in 50 m m glycine buffer at 30 °C. At pH 7.5 and above, this enzyme catalyzes significant rates of glucose oxidation at physiologically meaningful concentrations of glucose. A steady-state analysis of glucose dehydrogenase kinetics is made at 30 °C (pH 8.0, 9.5, and 10) and 37 °C (pH 7.5 and 9.5). At pH 9.5 and 37 °C, K ia 's for NAD and NADP are similar (5–6 μ m ) and the K b 's for glucose and glucose 6-phosphate are 4.2 m m and 25.4 μ m , respectively. At pH 7.5 and 37 °C, the K ia 's for NAD and NADP are 1.2 and 3–4 μ m , respectively, while the K b values for glucose and glucose 6-phosphate are 4.8 m m and 3.0 μ m . There is no apparent product inhibition by NADH or NADPH. Similarly, the glucose-NAD reaction is not inhibited by either glucose 6-phosphate or by 1,5-gluconolactone. An overall model is presented for the role of glucose dehydrogenase based upon kinetic evidence. Glucose dehydrogenase is seen as a multifunctional protein involved in several catabolic sugar pathways in the endoplasmic reticulum.

Glucose-6-phosphate dehydrogenase has been purified 1000-fold from pig liver. This enzyme exists as an active dimer of molecular weight 133,000 and an inactive monomer of molecular weight 67,500. The pH of maximum activity is 8.5 and the ionic strength maximum is 0.1 to 0.5 M. Glucose-6-phosphate dehydrogenase is highly specific for NADP+ and glucose 6-phosphate. Apparent Km values of 3.6 muM and 5.4 muM were obtained for glucose 6-phosphate and NADP+. This enzyme is located almost entirely within the soluble portion of the cellular cytoplasm.