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The ab initio structures and energies of a series of rotamers of 1-nitro-2-propanol have been calculated at the HF/6-31G(d) level. Correlation has been included at the MP2/6-31G(d)//HF/6-31G(d) level. The PES surface for rotation of the OH about a dihedral angle theta confirms the formation of an intramolecular hydrogen bond in the monomer which is stabilized by 5.4 kcal/mol at the MP2/6-31G(d)//HF/6-31G(d) level. Dimer formation via double intermolecular hydrogen bonds was found to be feasible by molecular mechanics and refined by ab initio computations. It is shown that a double intermolecular hydrogen bonded structure is stabilized by 12.9 kcal/mol relative to the monomer molecules at the HF/3-21G(d) level

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.

DMSO and TCNE form a strong 1:1 charge‐transfer complex (K25°C=95.4) with an absorption band located at λ=3720 A. The high enthalpy of complex formation (−11.3 kcal/mole) leads to an appreciably populated triplet state during complex formation. The EPR signal of the TCNE anion arises, reaches a maximum, and then follows second‐order decay kinetics. The initial intensity of the EPR signal is proportional to the original TCNE concentration in DMSO. A molecular mechanism for the equilibrium reaction between TCNE and DMSO is proposed.

The fluorescence characteristics of hematoporphyrin have been studied at 26°C as a function of concentration and pH. The pk values are 4.7 and 8.2 as determined in water and water-ethanol mixtures. The fluorescence spectrum consists of a peak at 625 nm and a second, less intense band at 675 nm. These emission bands are assigned to the Qy (0 → 0) and Qx(0 → 0) states. The four absorption bands in the visible region are assigned to π → π* transitions as follows: 502 nm, (1 ← 0); 536 nm, Qy (0 ← 0); 557 nm, Qx (1 ← 0); and 605 nm, Qx (0 ← 0). Corresponding excitation wavelengths include 505,540,570, and 620 nm. Polarized fluorescence is used to establish molecular association as a function of concentration. The polarization factor is used to verify the change from D2hto D4hsymmetry as a function of pH.

Porcine liver beta-D-glucose dehydrogenase has been isolated using Triton X-114 to release it from the endoplasmic reticulum. The purified enzyme contains a limited amount (1.7%) of lipid material, including cholesterol, fatty acids, mono and diglycerides, phosphatidylcholine, phosphatidylethanolamine, and cholesterol esters. This enzyme is a tetrameric protein containing an extensive number of hydrophobic residues. This form of glucose dehydrogenase is capable of turning over both beta-D-glucose and alpha-D-glucose-6-phosphate in vivo as indicated from a steady state kinetic analysis at 37 degrees C.

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.

We investigated the effect of disulfiram (Antabuse) on the activity of alcohol dehydrogenase (EC 1.1.1.1) in vitro. We observed a time-dependent inhibition of this dehydrogenase by disulfiram and diethyldithiocarbamate similar to that obtained for aldehyde dehydrogenase (EC 1.2.1.3). These results suggest a possible explanation for various side effects observed in the clinical use of Antabuse.

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.

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.

Porcine liver beta-D-glucose dehydrogenase, a multi-functional protein, has been purified to apparent homogeneity. The enzyme has been separated from the endoplasmic reticulum using Triton X-114 and further purified using NAD to release glucose dehydrogenase from a NADP-linked sepharose column. The purified enzyme is capable of producing both NADH and NADPH in vivo as indicated by kinetic studies.

In this experimental procedure, pyridoxamine is used to investigate some of the enzyme complexes formed by glucose dehydrogenase.

The interaction between magnesium and haematoporphyrin has been studied spectrophotometricaly as a function of concentration, pH and temperature and the existence of a strong 1 : 1 complex has been established. The Benesi-Hildebrand equation was used to determine Kc’s at various wavelengths and temperatures, at pH’s of 7.4, 8.2, and 9.0. The enthalpy of complex formation is seen to change from +9.78 kcal/mol to −9.50 kcal/mol as the pH changes from 8.2 to 9.0. This change may be a reflection of the change in the various ligands which coordinate to the magnesium ion in addition to the porphyrin ring.

A metal analysis of bovine hepatic gluconolactonase indicates the presence of at least 1 atom of tightly bound zinc per enzyme subunit in this hexameric protein. Other divalent metals are present in lesser quantities and are subject to removal by EDTA. Activation energies for the manganese and magnesium catalyzed reactions are 5.3 and 11.0 kcal/mole, respectively.

The interaction between magnesium and haematoporphyrin has been studied spectrophotometricaly as a function of concentration, pH and temperature and the existence of a strong 1 : 1 complex has been established. The Benesi-Hildebrand equation was used to determine Kc’s at various wavelengths and temperatures, at pH’s of 7.4, 8.2, and 9.0. The enthalpy of complex formation is seen to change from +9.78 kcal/mol to −9.50 kcal/mol as the pH changes from 8.2 to 9.0. This change may be a reflection of the change in the various ligands which coordinate to the magnesium ion in addition to the porphyrin ring.