James S. Franzen

Pyridine nucleotide-linked four-electron transfer dehydrogenases

Abstract Three types of four-electron transfer, pyridine nucleotide-linked dehydrogenases are known: β-methyl-β-hydroxyglutaryl CoA reductase, histidinol dehydrogenase, and nucleoside diphosphate sugar dehydrogenases. Although the reactions catalysed by the latter two classes of enzyme are very similar, the enzymes are structurally different.

Induced versus pre-existing asymmetry models for the half-of-the-sites reactivity effect in bovine liver uridine diphosphoglucose dehydrogenase

Half-of-the-sites reactivity of the catalytic site thiol groups of UDPglucose dehydrogenase (UDPglucose:NAD+ 6-oxidoreductase, EC 1.1.1.22) can be ascribed either to the induction of conformational asymmetry following derivatization of one half of the subunits or to intrinsic conformational differences in the subunits of the native enzyme. If the half-sites reactivity behavior is due to induction effects, the magnitude of the induction could be expected to depend on the nature of the covalent modification. On the other hand, if the half-sites reactivity behavior is due to pre-existing asymmetry and there is no communication between catalytic centers, the properties of unmodified sub-units should be independent of the nature of the covalent derivative introduced on the modified subunits. According to the induced asymmetry hypothesis, the catalytic activity of half-sites modified enzyme might be different for different covalent modifications, whereas for the rigid pre-existing asymmetry hypothesis the catalytic activity of half-sites modified enzyme should be the same regardless of the modifying group. During the course of catalytic site thiol group modification by a number of thiol specific reagents, the loss of enzyme activity was equivalent to the degree of modification for most of the reagents employed. However, with iodoacetate and 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid, half-sites modification of UDPglucose dehydrogenase reduced catalytic activity by 58 and 78%, respectively, of the initial activity. These observations are consistent with a model in which there is communication between catalytic sites. Electron microscopy shows that the six subunits of UDPglucose dehydrogenase are arranged as a hexagonal planar ensemble.

Oxidized triphosphopyridine nucleotide specific isocitrate dehydrogenase from Azotobacter vinelandii: III. The path of hydrogen in the enzymatic reaction

Abstract Isocitrate dehydrogenase from Azotobacter vinelandii transfers hydrogen from threo - d 8 -isocitrate to the A side of TPN + . The decarboxylation of threo d 8 isocitrate to 2-ketoglutarate occurs with retention of configuration. The enzyme catalyzes the labilization of tritium from [3-T]2-ketoglutarate in the presence of Mg 2+ and TPNH which suggests an ordered addition of substrates in the reductive carboxylation of 2-ketoglutarate to isocitrate.

UDP-glucose dehydrogenase: Substrate binding stoichiometry and affinity

Abstract The direct binding of UDP-glucose and NAD+ to bovine liver UDP-glucose dehydrogenase has been measured by equilibrium dialysis and differential fluorescence. At saturation the hexameric enzyme binds only three molecules each of UDP-glucose and NAD+. The binding of NAD+ is virtually characteristic of that for noninteracting identical sites with a binding constant of about 0.47 × 104. UDP-Glucose, however, binds more avidly than NAD+ and exhibits negative cooperativity characterized by unrestricted Adair constants of 16.1, 3.7, and 0.37, all × 104.

Binding ratios by fluorescence with self-compensation for optical inner filter effects

Abstract A simple procedure which self-compensates for optical inner filter effects has been developed for measuring the binding of ligands to macro-molecules by fluorescence measurements in moderately optically dense solutions. Correction for light source fluctuations during the course of measurement is also automatically achieved, provided the fluctuations are not too frequent, i.e., not greater than one every minute. The method is applicable both to systems involving fluorescing ligans which undergo a quantum efficiency change upon binding and to systems in which the macromolecule contains chromophores whose quantum efficiencies change upon ligand association. Examples of the use of the method with each type of system are presented in the text. The necessary fluorescence parameters are measurable with standard widely available commercial instrumentation. The general approach applied here to single-site proteins can, in principle, be extended to multisite systems as well.

Special effects of UDP-sugar binding to bovine liver uridine diphosphoglucose dehydrogenase

The binding of NADH to uridine diphosphate glucose dehydrogenase has been examined by equilibrium dialysis. There is an absolute requirement for the presence of UDP-glucose for the binding of NADH. Other analogs such as UDPxylose, UDPgalactose and UDPglucuronic acid cannot replace UDPglucose as an effector of NADH binding. UDPxylose competes with UDPglucose for the UDP-sugar-binding site, and in so doing releases the bound NADH. The binding of NADH to UDPglucose dehydrogenase in the presence of UDPglucose reaches a saturation limit of 3 mol NADH bound per enzyme hexamer, and displays positive cooperativity, Hill number = 1.34. The effects of UDP-sugars on the fluorescence of UDPglucose dehydrogenase derivatized at the catalytic sites with a fluorophore have also been studied. Two classes of UDPxylose-binding site have been detected. One class has high affinity (Kdiss = 3 microM, determined by equilibrium dialysis) but does not affect fluorophore fluorescence, and the other has lower affinity (Kdiss = 120 microM) and leads to red-shifted fluorescence quenching, presumably by effecting exposure of the fluorophore to solvent. The high-affinity sites are identified as the UDP-sugar subsites of the underivatized catalytic sites, and the low-affinity sites as UDP-sugar subsites of the fluorophore-labeled catalytic sites.

Binding studies with bovine liver udp-d-glucose dehydrogenase☆

Abstract The direct binding of uridine 5′-(α- d -glucopyranosyluronic acid pyrophosphate) (UDP- d -glucuronic acid) and of uridine 5′-(α- d -xylopyranosyl pyrophosphate) (UDP- d -xylose) to bovine liver UDP- d -glucose:NAD oxidoreductase (EC 1.1.1.22) has been measured by equilibrium dialysis. At saturation, the hexameric enzyme binds six molecules of UDP- d -glucuronic acid to noninteracting sites. UDP- d -xylose binds to 2 distinct classes of sites, each class binding six molecules of ligand. UDP- d -xylose is able to displace either UDP- d -glucose or UDP- d -glucuronic acid and UDP- d -glucose is able to displace UDP- d -glucuronic acid from the enzyme. It is proposed that the enzyme displays half-of-the-sites reactivity toward both substrate (UDP- d -glucose) and cosubstrate (NAD) and all-of-the-sites reactivity toward UDP- d -glucuronic acid. UDP- d -xylose is considered to bind cooperatively with high affinity to six regulatory sites and independently with lower affinity to six catalytic sites on the enzyme. Active-enzyme centrifugation studies show that UDP- d -glucose: NAD oxidoreductase is hexameric at concentrations corresponding to those used in steady-state kinetic measurements.

Reversible inactivation of Azotobacter vinelandii TPN+ -isocitrate dehydrogenase by the formation of an intramolecular disulfide bridge.

Abstract A method has been described for the formation of a modified form of isocitrate dehydrogenase which contains a single disulfide bridge. The modified enzyme is inactive and exhibits a decrease in a-helix content. The modified enzyme is inactive but catalytic activity can be restored by cleavage of the disulfide bridge. The original a-helix content is not restored upon reduction.

Oxidation Numbers in the Study of Metabolism.

The calculation and use of oxidation numbers in the study of metabolic reactions are discussed for normal oxidations (alcohol dehydrogenase and the NAD+/NADH couple, propanediol dehydratase) and for enzymatic reactions with a “hidden” redox component (transamination, the coupled conversion of ethylamine to ethanol, and the biomethylation of arsenic and selenium).

Influence of effectors on the rate of reaction of reduced ribonucleoside triphosphate reductase with N-ethylmaleimide.

Abstract Ribonucleoside triphosphate reductase from Lactobacillus leichmannii , after reduction by exposure to dithiothreitol, has been alkylated with N -ethylmaleimide. Under conditions where the unreduced enzyme does not incorporate N -ethylmaleimide residues, the reduced enzyme is rapidly alkylated to the extent of one N -ethylmaleimide per molecule of enzyme. Loss of enzyme activity parallels the incorporation of N -ethylmaleimide. The value of the second-order rate constant for the alkylation at 0 °C of the reduced enzyme is influenced by the presence of some of the effectors of the enzyme, e.g., dATP at 200 μ m reduces this parameter from 0.61 to 0.33 m m −1 min −1 . The addition of coenzyme B 12 did not significantly affect the rate of alkylation of the reduced enzyme nor did it change the rate of alkylation of the dATP-reduced enzyme complex. Reduced enzyme, freed of dithiol, was shown to be unable to convert CTP stoichiometrically to dCTP when all of the usual enzyme assay components, except the dithiol, were present, nor did addition of CTP to the otherwise complete mixture decrease the level of N -ethylmaleimide-reactive thiol. However, the subsequent addition of dithiol was found to result in essentially complete reduction of CTP to dCTP. Hence, although reduction of the enzyme is probably required to generate an active form of the enzyme, the reduced enzyme does not appear to be capable of transferring its reducing equivalents stoichiometrically to the substrate to form dCTP from CTP. These results are discussed in terms of the mechanism of action of this enzyme.