Dihydronicotinamide Ring Research Articles

Properties of crystalline leucine dehydrogenase from Bacillus sphaericus

The distribution of bacterial leucine dehydrogenase (L-leucine:NAD+ oxidoreductase, deaminating, EC 1.4.1.9) was investigated, and Bacillus sphaericus (IFO 3525) was found to have the highest activity of the enzyme. Leucine dehydrogenase, which was purified to homogeneity and crystallized from B. sphaericus, has a molecular weight of about 245,000 and consists of six identical subunits (Mr = 41,000). The enzyme catalyzes the oxidative deamination of L-leucine, L-valine, L-isoleucine, L-norvaline, L-alpha-aminobutyrate, and L-norleucine, and the reductive amination of their keto analogues. The enzyme requires NAD+ as a cofactor, which cannot be replaced by NADP+. D-Enantiomers of the substrate amino acids inhibit competitively the oxidation of L-leucine. The enzyme activity is significantly reduced by both sulfhydryl reagents and pyridoxal 5'-phosphate. Purine and pyrimidine bases, nucleosides and nucleotides have no effect on the enzyme activity. Initial velocity and product inhibition studies show that the reductive amination proceeds through a sequential ordered ternary-binary mechanism. NADH binds first to the enzyme followed by alpha-ketoisocaproate and ammonia, and the products are released in the order of L-leucine and NAD+. The Michaelis constants are as follows: L-leucine (1 mM), NAD+ (0.39 mM), NADH (35 micrometer), alpha-ketoisocaproate (0.31 mM), and ammonia (0.2 M). The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH is exclusively transferred to the substrate; the enzyme is B-stereospecific.

A transition state analog for two pyruvate metabolizing enzymes, lactate dehydrogenase and alanine dehydrogenase

The synthesis of 5-(2-oxalylethyl)-NADH, a reduced nicotinamide adenine dinucleotide (NADH) derivate with pyruvate covalently attached to the 5 position of the dihydronicotinamide ring over an additional methylene group has been described previously (Trommer, W.E., Blume, H., and Kapmeyer, H. (1976) Justus Liebigs Ann. Chem., 848). In the presence of lactate dehydrogenase, the dihydropyridine ring of this coenzyme-substrate analogue is oxidized and the carbonyl function of the side chain is reduced to the corresponding L-hydroxy derivative with a maximum velocity of 1/3000 of the natural reaction. This reaction is intramolecular as shown by competition experiments with pyruvate. 5-(2-oxalylethyl)-NADH (pyr-NADH) appears to be a true transition state analogue, proving its postulated structure. Pyr-NADH is high specific for this enzyme as demonstrated by the facts that (1) D-lactate dehydrogenase does not catalyze the intramolecular redox reaction, although the substrate moiety of pyr-NADH is reduced in the presence of NADH; (2) when tested with malate dehydrogenase, alcohol dehydrogenase, glyceraldehyde phosphate dehydrogenase,glycerate dehydrogenase, and glycerol dehydrogenase pyr-NADH is not even oxidized in the presence of the corresponding substrates. However, a great similarity between the transition states of the reduction of pyruvate catalyzed by lactate dehydrogenase and alanine dehydrogenase could be shown. Alanine dehydrogenase catalyzes the intramolecular redox reaction as well. In the presence of ammonium ions, pyr-NADH is transformed to 5-(3-carboxyl-3-aminopropyl)-NAD+.

The alpha beta epimerization of reduced nicotinamide adenine dinucleotide

The proton magnetic resonance spectra of the dihydronicotinamide ring of αNADH 3 3 αNAD + and αNADH, the nicotinamide adenine dinucleotides in which the nicotinamide-ribosidic linkage has an α configuration; (4D)αNAD +, αNAD + with a deuterium label at the C4 position; αNADD B, βNADD B, α and βNADH with a deuterium label in the C4B position of the dihydronicotinamide ring; Y.ADH, yeast alcohol dehydrogenase. and the nicotinamide ring of αNAD + are reported and the proton absorptions assigned. The absolute assignment of the C4 methylene protons of αNADH is based on the generation of specifically deuterium-labeled (pro-S) B-deuterio-αNADH from enzymatically prepared B-deuterio-βNADH. The C4 proton absorption of αNAD + is assigned by oxidation of B-deuterio-αNADH by the A specific, yeast alcohol dehydrogenase to yield 4-deuterio-αNAD +. The epimerization of either αNADH or βNADH yields an equilibrium ratio of approximately 9:1 βNADH to αNADH. The rate of epimerization of αNADH to βNADH at 38 °C in 0.05, pH 7.5, phosphate buffer is 3.1 × 10 −3 min −1, corresponding to a half-life of 4 hr. Four related dehydrogenases, yeast and horse liver alcohol dehydrogenase and chicken M 4 and H 4 lactate dehydrogenase, are shown to oxidize αNADH to αNAD + at rates three to four orders of magnitude slower than for βNADH. By using specifically labeled B-deuterio-αNADH the enzymatic oxidation by yeast alcohol dehydrogenase has been shown to occur with the identical stereospecificity as the oxidation of βNADH. The nonenzymatic epimerization of αNADH to βNADH and the enzymatic oxidation αNADH are discussed as a possible source of αNAD + in vivo.

Studies on the biosynthesis of cholesterol. XVII. The asymmetric synthesis of a symmetrical molecule

The differentiation between two identical chemical groups in the Ca'a"bd molecule in enzymic reactions is discussed first. It is then shown that squalene biosynthesized from two molecules of [1-D 2 ]farnesyl pyrophosphate is asymmetrically labelled at one of its central methylene carbon atoms, one of the deuterium atoms at C-l of one of the two farnesyl residues having been replaced stereospecifically by H B of reduced nicotinamide adenine dinucleotide phosphate. The trideuterio succinic acid obtained by ozonolysis from such squalene is shown to be dextrorotatory and to have the S absolute configuration. The steric position of H A and of H B at position 4 of the dihydronicotinamide ring in NADH and NADPH, transferred from substrates to coenzyme by ‘A’- and ‘B’-specific dehydrogenases respectively, is deduced. This was achieved by making a correlation between the laevorotatory 2 R -[2-D 1 ]succinic acid (made chemically from enzymically generated 2 S -3 R -[3-D 1 ]malic acid) and the [2-D 1 ]succinic acids derived by chemical degradation from positions 3, 4, 5 and 6 of the dihydronicotinamide ring of ‘A’-deuterio and ‘B’-deuterio NADH. The absolute configuration of C-4 of the dihydronicotinamide ring in ‘A’-deuterio NADH is R and in the ‘B’-deuterio NADH it is S .