Mechanism of the enzymatic reaction catalyzed by uridine diphosphate glucose dehydrogenase. The chemical synthesis of uridine diphosphate glucose-6-H3 and its oxidation by uridine diphosphate glucose dehydrogenase]

The synthesis of UDP-glucose-6-s-H was performed through condensation of alpha-D-glucopyranosyl phosphate-6-3-H and uridine 5'-phosphomorpholidate. Enzymic oxidation of UDP-glucose-6-3-H with calf liver UDP-glucose dehydrogenase was found to proceed with direct transfer of the hydrogen from C-6 of UDP-glucose onto NAD.     

Stereospecificity of hydrogen transfer in the saccharopine dehydrogenase reaction.

The stereospecificity of hydrogen transfer in the synthesis of saccharopine from alpha-ketoglutarate and L-lysine catalyzed by saccharopine dehydrogenase (N5-(1,3-dicarboxypropyl)-L-lysine: NAD oxidoreductase (L-lysine-forming), EC 1.5.1.7) was examined by using [4A-3H]- and [4B-3H]NADH. The enzyme showed the A-stereospecificity. The NMR analysis of the saccharopine prepared with [4"A-2H]NADH revealed that the label was incorporated into the C-2 of the glutaryl moiety.     

Stereochemistry of the hydrogen transfer to NADP catalyzed by D-galactose dehydrogenase from Pseudomonas fluorescens.

The stereochemistry of the hydrogen transfer to NADP catalyzed by D-galactose dehydrogenase (EC 1.1.1.48) from P. fluorescens was investigated. The label at C-1 of D-[1-³H] galactose was enzymatically transferred to NADP and the resulting [4-³H]NADPH was isolated and its stereo-chemistry at C-4 investigated. It was found that the label was exclusively located at the 4(S) position in NADPH which calls for classification as a B-enzyme. The correlation of this finding with tentative classification rules of NAD(P)-linked dehydrogenases in regard to their stereo-chemistry of hydrogen transfer to the coenzyme is discussed.     

Stereospecificity of hydrogen transfer by phosphoglycerate dehydrogenase.

Phosphoglycerate dehydrogenase (EC 1.1.1.95) has been shown to be A site specific in its hydrogen transfer capacity unlike other dehydrogenases which use phosphorylated substrates. The experiments have been carried out using a coupled assay system with yeast alcohol dehydrogenase. The specific activity measurements of the reaction products indicate the possible influence of an isotope effect on this system.     

Redox-elimination reaction catalyzed by yeast alcohol dehydrogenase

Yeast alcohol dehydrogenase (EC 1.1.1.1) catalyzed reduction of N,N-dimethyl-4-nitrosoaniline by NADH. The stoichiometry of reaction, steady-state kinetic parameters, and the pH-profile for this reaction were estimated. On that basis, the minimal mechanism of the above reaction was postulated.     

Stereochemistry of the hydrogen transfer to NAD catalyzed by (S)alanine dehydrogenase from Bacillus subtilis.

The stereochemistry of the hydrogen transfer to NAD catalyzed by (S)alanine dehydrogenase [ (S)alanine: NAD oxidoreductase (EC 1.4.1.1) ] from B. subtilis was investigated. The label at C-2 of (S) [2,3--3H] alanine was enzymatically transferred to NAD, and the [4--3H]NADH produced isolated and the stereochemistry at C-4 investigated. It was found that the label was exclusively located at the (R) position which indicates that (S)alanine dehydrogenase is an A-type enzyme. This result was confirmed in an alternate way by reducing enzymatically [4--3H]NAD with non labeled (S)alanine and (S)alanine dehydrogenase and investigating the stereochemistry of the ]4--3H]NADH produced. As expected, the label was now exclusively located at the (S) position. This proves that (S)alanine dehydrogenase isolated from B. subtilis should be classified as an A-enzyme with regard to the stereochemistry of the hydrogen transfer to NAD.     

The effect of anions on the reaction catalyzed by lupine-nodule glutamate dehydrogenase

The kinetics of the reductive amination reaction of lupine-nodule glutamate dehydrogenase (l-glutamate:NAD oxidoreductase (deaminating), EC 1.4.1.2) were found to vary with the identity of the ammonium salt which was used as a substrate. Normal Michaelis-Menten kinetics were obtained with (NH₄)₂SO₄ but when NH₄Cl or NH₄-acetate was varied apparent substrate inhibition was observed. Linear double-reciprocal plots were obtained with NH₄Cl and NH₄-acetate, however, if the concentration of Cl^? or acetate was maintained constant by adding KCl or K-acetate. Chloride and acetate were subsequently found to cause linear noncompetitive inhibition with respect to NH₄⁺ and the apparent substrate inhibition by NH₄Cl and NH₄-acetate can be explained as the result varying a substrate and a noncompetitive inhibitor in constant ratio. Other anions were also found to be inhibitors of the glutamate dehydrogenase reaction; I^? caused parabolic noncompetitive inhibition with respect to NH₄⁺ and NO₃^? caused slope-parabolic noncompetitive inhibition with respect to all three substrates of the reductive amination reaction. For the oxidation deamination reaction, Cl^? was a linear competitive inhibitor with respect to both NAD and l-glutamate whereas NO₃^? caused parabolic competitive inhibition with respect to these reactants. To explain the results, it is proposed that anions bind to an allosteric site and cause a change in some of the rate constants of the reaction. Specifically, the results are consistent with anions causing decreases in the rates of association of NADH and 2-oxoglutarate with the enzyme and an increase in the rate of dissociation of NAD.     

Chirality of the hydrogen transfer to the coenzyme catalyzed by ribitol dehydrogenase from Klebsiella pneumoniae and D-mannitol 1-phosphate dehydrogenase from Escherichia coli.

The stereochemistry of the hydrogen transfer to NAD catalyzed by ribitol dehydrogenase (ribitol:NAD 2-oxidoreductase, EC 1.1.1.56) from Klebsiella pneumoniae and D-mannitol-1-phosphate dehydrogenase (D-mannitol-1-phosphate:NAD 2-oxidoreductase, EC 1.1.1.17) from Escherichia coli was investigated. [4-3H]NAD was enzymatically reduced with nonlabelled ribitol in the presence of ribitol dehydrogenase and with nonlabelled D-mannitol 1-phosphate and D-mannitol 1-phosphate dehydrogenase, respectively. In both cases the [4-3H]-NADH produced was isolated and the chirality at the C-4 position determined. It was found that after the transfer of hydride, the label was in both reactions exclusively confined to the (4R) position of the newly formed [4-3H]NADH. In order to explain these results, the hydrogen transferred from the nonlabelled substrates to [4-3H]NAD must have entered the (4S) position of the nicotinamide ring. These data indicate for both investigated inducible dehydrogenases a classification as B or (S) type enzymes. Ribitol also can be dehydrogenated by the constitutive A-type L-iditol dehydrogenase (L-iditol:NAD 5-oxidoreductase, EC 1.1.1.14) from sheep liver. When L-iditol dehydrogenase utilizes ribitol as hydrogen donor, the same A-type classification for this oxidoreductase, as expected, holds true. For the first time, opposite chirality of hydrogen transfer to NAD in one organic reaction--ribitol + NAD = D-ribu + NADH + H--is observed when two different dehydrogenases, the inducible ribitol dehydrogenase from K. pneumoniae and the constitutive L-iditol dehydrogenase from sheep liver, are used as enzymes. This result contradicts the previous generalization that the chirality of hydrogen transfer to the coenzyme for the same reaction is independent of the source of the catalyzing enzyme.