Miguel A. Alizade

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.

Stereochemistry of the l-iditol dehydrogenase catalyzed hydrogen transfer to NAD

Abstract The stereochemistry of the hydrogen transfer to NAD catalyzed by l -iditol dehydrogenase from sheep liver (EC 1.1.1.14) was investigated. [4-3H]NAD was enzymatically reduced with non-labeled d -sorbitol and the position of the label at C4 of the isolated [4-3H]NADH was determined. The label was found to be exclusively confined to the B-position which suggests that the transferred hydrogen from d -sorbitol must have entered into the A-position of the NADH produced. These data imply that l -iditol dehydrogenase from sheep liver is an A-enzyme.

The stereospecificity of the hydrogen transfer from (R)-carnitine to NAD, catalyzed by (R)-carnitine dehydrogenase from Pseudomonas aeruginosa

Abstract The transfer of hydrogen from the C 3 position of ( R )-carnitine to NAD, catalyzed by ( R )-carnitine dehydrogenase from Pseudomonas aeruginosa was investigated. This transfer was shown to be a direct one involving the B-position at C 4 of the pyridine ring of the nucleotide. In confirmation of this finding, the transfer of hydrogen from ( R )-carnitine to [4- 3 H]NAD resulted in NADH with the label confined to the A-position. These experiments show that ( R )-carnitine dehydrogenase should be classified as a B-enzyme.

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

Abstract 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.

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.

A rapid method of separation and determination of quaternary ammonium compounds involved in carnitine metabolism.

Abstract The rapid separation of several quaternary ammonium compounds involved in carnitine metabolism is reported. Two convenient methods based on spectrophotometry and liquid scintillation counting are employed to determine the eluting compounds. The application of the techniques to biological determinations is discussed.

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.

Abstract Stereospecific, Hydrogen Labeled The preparation of (R) and (S) [2-3H] lactate as well as (S) [2-3H] glutam ate via the coupled exchange reaction catalyzed by NAD linked dehydrogenases and NADH : lipoam ide 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 w ith higher specific radioactivity are obtained from positionally [3H] labeled racem ic m ixtures prepared by chemical reductions with [3H] labeled hydrides. The tritium content of one of the enantiom eres 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, glutam ate and (R) and (S) lactate.

Analytical application of the stereospecific hydrogen exchange reaction between (R)-carnitine and water, catalyzed by (R)-carnitine dehydrogenase and α-lipoamide dehydrogenase (diaphorase)

Abstract A stereospecific hydrogen exchange between tritiated water and the hydrogen at C 3 of (R)-carnitine takes place under the coupled catalyses of (R)-carnitine dehydrogenase from Pseudomonas aeruginosa and α-lipoamide dehydrogenase (diaphorase) from pig heart. This exchange reaction can be used to synthesize (R)-(3- 3 H) -carnitine. The amount of tritium released from the C 3 position of (R)-(3- 3 H) -carnitine into water is decreased proportionally by the addition of non labelled (R)-carnitine, making possible a new sensitive assay for (R)-carnitine.

Chirality of the Hydrogen Transfer to NAD Catalyzed by (3R)Hydroxybutyrate Dehydro­genase from Pseudomonas lemoignei

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

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

Abstract An isotope effect of the dehydrogenation of (R) Carnitine [(R) 3-hydroxy-4-trimethylamino-butyric acid hydrochloride] catalyzed by (R) carnitine dehydrogenase [(R) carnitine: NAD oxido-reductase 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.