Chirality of the Hydrogen transfer to NAD catalyzed by (3R) hydroxybutyrate dehydrogenase from Pseudomonas lemoignei.

The chirality of the hydrogen transfer from (3R) hydroxybutyrate to NAD catalyzed by (3R)hydroxybutyrate dehydrogenase (E.C. 1.1.1.30, D-3-hydroxybutyrate : NAD oxidoreductase) from Pseudomonas lemoignei was investigated. [4(-3)H] NAD was enzymatically reduced to (4R) [4(-3)H]NADH with (3RS) hydroxybutyrate. This observation was confirmed since NAD could be reduced to (4S) [4(-3)H] NADH with (3RS) [3(-3)H] hydroxybutyrate and (3R) hydroxybutyrate dehydrogenase. From these experiments it can be concluded that (3R) hydroxybutyrate dehydrogenase from P. lemoignei should be classified as an B or (S) type dehydrogenase.

Chirality of the hydrogen transfer to NAD catalyzed by myo-inositol dehydrogenase from Klebsiella pneumoniae.

Chirality, Hydrogen Transfer, myo-Inositol Dehydrogenase. The chirality of the hydrogen transfer to NAD catalyzed by myo-inositol dehydrogenase (myo-Inositol: NAD 2-oxydoreductase, EC 1.1.1.18) from Klebsiella pneumoniae (formerly classified taxonomically as Aerobacter aerogenes or Kleb siella aerogenes) was investigated. [4-3H] NAD was enzymatically reduced to [4-3H] NADH with non-labeled myoinositol and myo-inositol dehydrogenase. The stereochemistry of the prochiral center at C4 of the NADH produced was determined. It was found that the label was exclusively located at the (4S) position of the produced NADH. Since the hydrogen transferred from non-labeled myo-inositol to [4-3H] NAD must have entered the opposite of (R) position, myo-inositol dehydrogenase from K. pneumoniae should be classified as an (R) or A-type enzyme with respect to the stereochemistry of the hydrogen transfer to 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.

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

The stereochemistry of the hydrogen transfer to NAD catalyzed by D-galactose dehydrogenase (E.C. 1.1.1.48) from P. fluorescens was investigated. The label at C-1 of D-[1--3H] galactose was enzymatically transferred to NAD and the resulting [4--3H]NADH was isolated and its stereochemistry at C-4 investigated. It was found that the label was exclusively located at the 4(S) position in NADH which calls for classification as a B-enzyme. This result was confirmed by an alternate approach in which [4--3H]NAD was reduced by D-galactose as catalyzed by D-galactose dehydrogenase. The sterochemistry at C-4 of the nicotinamide ring would then have to opposite to that in the first experiment. As expected, the label was now exclusively located in the 4(R) position, again confirming the B-calssification of the enzyme.

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.

Determination of the isotope effect of the enzymatic oxidation of (R)Carnitine by displacement of the equilibrium via mass action.

An isotope effect of the dehydrogenation of (R) Carnitine [(R) 3-hydroxy-4-trimethylaminobutyric acid hydrochloride] catalyzed by (R) carnitine dehydrogenase [(R) carnitine: NAD oxidoreductase E.C. 1.1.1.108] from Pseudomonas aeruginosa was measured at different temperatures. It was found that k1H/k3H does not vary greatly with changes of temperature. The value of 3 for k1H/k3H measured at small initial conversions strongly indicated that the rate limiting step of the oxidation of (R) carnitine is the cleavage of the C-H bond at C3.

Biochemical synthesis of stereospecifically hydrogen labeled compounds on a preparative scale, VI1-3 Synthesis of further substrates of NAD(P)-linked dehydrogenases of high specific tritium content.

The preparation of (R) and (S) [2(-3)H]lactate as well as (S) [2(-3)H] glutamate via the coupled exchange reaction catalyzed by NAD linked dehydrogenases and NADH: lipoamide oxidoreductase (diaphorase) is described. The specific radioactivity of the hydrogen ions of the 3HOH/H2O can be obtained in the substrates (100% exchange) if equilibrium isotope effects are disregarded. By the exchange procedure substrates with higher specific radioactivity are obtained from positionally [3H]labeled racemic mixtures prepared by chemical reductions with [3H]labeled hydrides. The tritium content of one of the enantiomeres is "washed out" into water. As examples are presented the preparation of (R) [2-3H] (S) [2-H]malate as well as the corresponding carnitine, glutamate and (R) and (S) lactate.