Regio- and stereospecificity of homogeneous 3 alpha-hydroxysteroid-dihydrodiol dehydrogenase for trans-dihydrodiol metabolites of polycyclic aromatic hydrocarbons

The homogeneous 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) of rat liver cytosol is indistinguishable from dihydrodiol dehydrogenase (trans-1,2-dihydrobenzene-1,2-diol dehydrogenase EC 1.3.1.20), Penning, T. M., Mukharji, I., Barrows, S., and Talalay, P. (1984) Biochem. J. 222, 601-611). Examination of the substrate specificity of the purified dehydrogenase for trans-dihydrodiol metabolites of polycyclic aromatic hydrocarbons indicates that the enzyme will catalyze the NAD(P)-dependent oxidation of trans-dihydrodiols of benzene, naphthalene, phenanthrene, chrysene, 5-methylchrysene, and benzo[a]pyrene under physiological conditions. Comparison of the utilization ratios Vmax/Km indicates that benzenedihydrodiol and the trans-1,2- and trans-7,8-dihydrodiols of 5-methylchrysene were most efficiently oxidized by the purified dehydrogenase, followed by the trans-7,8-dihydrodiol of benzo[a]pyrene and the trans-1,2-dihydrodiols of phenanthrene, chrysene, and naphthalene. The purified enzyme appears to display rigid regio-selectivity, since it will readily oxidize non-K-region trans-dihydrodiols but will not oxidize the K-region trans-dihydrodiols of phenanthrene and benzo[a]pyrene. The stereochemical course of enzymatic dehydrogenation was investigated by circular dichroism spectrometry. For the trans-1,2-dihydrodiols of benzene, naphthalene, phenanthrene, chrysene, and 5-methylchrysene, the dehydrogenase preferentially oxidized the (+)-[S,S]-isomer. Apparent inversion of this stereochemical preference occurred with the trans-7,8-dihydrodiol of 5-methylchrysene, as the (-)-enantiomer was preferentially oxidized. No change in the sign of the Cotton Effect was observed following oxidation of the racemic trans-7,8-dihydrodiol of benzo[a]pyrene, suggesting that both stereoisomers of this compound were substrates. Large-scale incubation of the [3H]-(+/-)-trans-7,8-dihydrodiol of benzo[a]pyrene with the purified dehydrogenase resulted in greater than 90% utilization of this potent proximate carcinogen, suggesting that the enzyme utilizes both the (-)-[R,R] and the (+)-[S,S]-stereoisomers, which confirms the circular dichroism result. These data show that dihydrodiol dehydrogenase displays the appropriate regio- and stereospecificity to catalyze the oxidation of both the major and minor non-K-region trans-dihydrodiols that arise from the microsomal metabolism of benzo[a]pyrene in vivo.     

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 (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.     

Kinetic properties of N-(2-carboxyethyl)-NAD(H) and poly(ethylene glycol)-bound NAD(H) for alcohol, lactate, malate and glyceraldehyde-3-phosphate dehydrogenase from different organisms

The steady-state kinetics of alcohol dehydrogenases (alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1 and alcohol:NADP⁺ oxidoreductase, EC 1.1.1.2), lactate dehydrogenases (l-lactate:NAD⁺ oxidoreductase, EC 1.1.1.27 and d-lactate:NAD⁺ oxidoreductase, EC 1.1.1.28), malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37), and glyceraldehyde-3-phosphate dehydrogenases [d-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase (phosphorylating), EC 1.2.1.12] from different sources (prokaryote and eukaryote, mesophilic and thermophilic organisms) have been studied using NAD(H), N⁶-(2-carboxyethyl)-NAD(H), and poly(ethylene glycol)-bound NAD(H) as coenzymes. The kinetic constants for NAD(H) were changed by carboxyethylation of the 6-amino group of the adenine ring and by conversion to macromolecular form. Enzymes from thermophilic bacteria showed especially high activities for the derivatives. The relative values of the maximum velocity (NAD = 1) of Thermus thermophilus malate dehydrogenase for N⁶-(2-carboxyethyl)-NAD and poly(ethylene glycol)-bound NAD were 5.7 and 1.9, respectively, and that of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase for poly(ethylene glycol)-bound NAD was 1.9.     

Amino acid sequence of the nucleotide-binding site of D-(-)-beta-hydroxybutyrate dehydrogenase labeled with arylazido-beta-[3-3H]alanylnicotinamide adenine dinucleotide

In the dark, arylazido-beta-alanylnicotinamide adenine dinucleotide (N3-NAD) can replace NAD as cofactor for D-(-)-beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria. When photoirradiated with visible light, N3-NAD forms a nitrene species that binds covalently to BDH and inhibits the enzyme. NAD(H) protects BDH against photolabeling and inhibition by N3-NAD [Yamaguchi, M., Chen, S., & Hatefi, Y. (1985) Biochemistry 24, 4912-4916]. In the present study, a tryptic peptide of purified BDH photolabeled with arylazido-beta-[3-3H] alanyl-NAD [( 3H]N3-NAD) was isolated and sequenced. The same tryptic peptide was also isolated from BDH not labeled with [3H]N3-NAD and sequenced. Both peptides indicated the sequence Met-Glu-Ser-Tyr-Cys-Thr-Ser-Gly-Ser-Thr-Asp-Thr-Ser-Pro-Val-Ile-Lys. The residue labeled with [3H]N3-NAD was Cys. This heptadecapeptide contains 14 uncharged residues and is marked by having in an undecapeptide segment 8 hydroxy amino acids located symmetrically around a central glycine.     

Regeneration of NAD(H) covalently bound to formate dehydrogenase with several second enzymes

Summary N ⁶-[N-(6-Aminohexyl)carbamoylmethyl]-NAD was covalently bound to formate dehydrogenase. The formate dehydrogenase-NAD complex, which contained 0.2 mol of reactive NAD moiety per subunit, functioned as an NAD(H)-regeneration system for a second coupled reaction involving one of the following enzymes; lactate, malate, alanine and leucine dehydrogenases, whose reductive reactions proceeded stoichiometrically in the absence of exogenous NAD.     

Enzymatic in situ analysis by 1H-NMR of the hydrogen transfer stereospecificity of NAD(P)+-dependent dehydrogenases

We have established a simple procedure for the in situ analysis of stereospecificity of an NAD(P)-dependent dehydrogenase for C-4 hydrogen transfer of NAD(P)H by means of glutamate racemase [EC 5.1.13] and glutamate dehydrogenase [EC 1.4.1.3]. Glutamate racemase inherently catalyzes the exchange of alpha-H of glutamate with 2H during racemization in 2H2O. When the reactions of glutamate racemase and glutamate dehydrogenase, which is pro-S specific for the C4-H transfer of NAD(P)H, are coupled in 2H2O, [4S-2H]-NAD(P)H is exclusively produced. Therefore, if 1H is fully retained at C-4 of NAD(P)+ after incubation of a reaction mixture containing both the enzymes and a dehydrogenase to be tested, the stereospecificity of the dehydrogenase is the same as that of glutamate dehydrogenase. When the C4-H of NAD(P)+ is exchanged with 2H, the enzyme to be examined is different from glutamate dehydrogenase in stereospecificity. Thus, we can readily determine the stereospecificity by 1H-NMR measurement of NAD(P)+ without isolation of the coenzymes and products.     

The ubiquitous aldehyde reductase (AKR1A1) oxidizes proximate carcinogen trans-dihydrodiols to o-quinones: potential role in polycyclic aromatic hydrocarbon activation

Polycyclic aromatic hydrocarbons (PAHs) are metabolized to trans-dihydrodiol proximate carcinogens by human epoxide hydrolase (EH) and CYP1A1. Human dihydrodiol dehydrogenase isoforms (AKR1C1-AKR1C4), members of the aldo-keto reductase (AKR) superfamily, activate trans-dihydrodiols by converting them to reactive and redox-active o-quinones. We now show that the constitutively and widely expressed human AKR, aldehyde reductase (AKR1A1), will oxidize potent proximate carcinogen trans-dihydrodiols to their corresponding o-quinones. cDNA encoding AKR1A1 was isolated from HepG2 cells, overexpressed in Escherichia coli, purified to homogeneity, and characterized. AKR1A1 oxidized the potent proximate carcinogen (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene with a higher utilization ratio (V(max)/K(m)) than any other human AKR. AKR1A1 also displayed a high V(max)/K(m) for the oxidation of 5-methylchrysene-7,8-diol, benz[a]anthracene-3,4-diol, 7-methylbenz[a]anthracene-3,4-diol, and 7,12-dimethylbenz[a]anthracene-3,4-diol. AKR1A1 displayed rigid regioselectivity by preferentially oxidizing non-K-region trans-dihydrodiols. The enzyme was stereoselective and oxidized 50% of each racemic PAH trans-dihydrodiol tested. The absolute stereochemistries of the reactions were assigned by circular dichroism spectrometry. AKR1A1 preferentially oxidized the metabolically relevant (-)-benzo[a]pyrene-7(R),8(R)-dihydrodiol. AKR1A1 also preferred (-)-benz[a]anthracene-3(R),4(R)-dihydrodiol, (+)-7-methylbenz[a]anthracene-3(S),4(S)-dihydrodiol, and (-)-7,12-dimethylbenz[a]anthracene-3(R),4(R)-dihydrodiol. The product of the AKR1A1-catalyzed oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene was trapped with 2-mercaptoethanol and characterized as a thioether conjugate of benzo[a]pyrene-7,8-dione by LC/MS. Multiple human tissue expression array analysis showed coexpression of AKR1A1, CYP1A1, and EH, indicating that trans-dihydrodiol substrates are formed in the same tissues in which AKR1A1 is expressed. The ability of this general metabolic enzyme to divert trans-dihydrodiols to o-quinones suggests that this pathway of PAH activation may be widespread in human tissues.     

Photoaffinity labeling of D-(-)-beta-hydroxybutyrate dehydrogenase by (arylazido)-beta-alanyl-substituted nicotinamide adenine dinucleotide

(Arylazido)-beta-alanyl-substituted nicotinamide adenine dinucleotide (N3-NAD) is a photosensitive analogue of NAD capable of photoinduced nitrene generation and insertion into a nearby molecule. In the dark, N3-NAD can replace NAD as a cosubstrate for the mitochondrial D-(-)-beta-hydroxybutyrate dehydrogenase (BDH). With purified, phospholipid-reconstituted BDH and NAD as the variable substrate, the apparent Km and Vmax values were respectively 0.25 mM and 62.5 mumol min-1 (mg of protein)-1. With N3-NAD as the variable substrate, these values were respectively 0.59 mM and 5 mumol min-1 (mg of protein)-1. Photoirradiation of BDH in the presence of N3-NAD resulted in irreversible inhibition of the enzyme and incorporation into the protein of radioactivity from tritiated N3-NAD. Photoirradiation of BDH plus or minus NAD in the absence or presence of (arylazido)-beta-alanine caused little or no inhibition. The photoinhibition of BDH in the presence of N3-NAD was prevented nearly completely by addition of NADH, NAD plus beta-hydroxybutyrate, or NAD plus 2-methylmalonate and partially by addition of NAD. Moreover, the presence of NADH prevented, and prior partial modification of BDH at the NAD(H)-protectable site by N-ethylmaleimide decreased, the incorporation of radioactivity into BDH from photoirradiated [3H]N3-NAD. The above results suggest that N3-NAD can be used for photoaffinity labeling of BDH at the active site.     

A kinetic study on the binding of monomeric and polymeric derivatives of NAD+ to yeast alcohol dehydrogenase

The binding to yeast alcohol dehydrogenase of NAD+ and its five derivatives (N6-[2-[N-[2-[N-(2-methacrylamidoethyl)carbamoyl]ethyl] carbamoyl]ethyl]-NAD (I), N6-[N-[2-[N-(2-methacrylamidoethyl) carbamoyl]ethyl]carbamoylmethyl]-NAD (II), copolymer of I with acrylamide (PA-I), copolymer of II with acrylamide (PA-II), and copolymer of I with N,N-dimethylacrylamide (PDMA-I] were studied statically and kinetically by the stopped-flow method by using the quenching of the enzyme fluorescence in the presence of pyrazole. Apparent dissociation constants and apparent rate constants were determined therefrom. It was concluded that (1) the N6-CH2CH2CO group (of I) is effective in making the derivative bind more strongly as well as faster than NAD+, while the N6-CH2CO group (of II) is not; and (2) the binding of the polymer derivatives of NAD+ to the enzyme is not essentially weaker and slower than that of native NAD+, but is even faster in some cases. The coenzymic activities of the above compounds were also determined with yeast alcohol dehydrogenase, pig heart malate dehydrogenase, and rabbit muscle lactate dehydrogenase.     

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.     

NAD recycling in the collagen membrane.

NAD recycling in the collagen membrane was investigated as follows: (1) Alcohol dehydrogenase and lactate dehydrogenase were co-immobilized in the collagen membrane and the rate of lactate production by immobilized enzymes was compared with that of free enzymes by using free NAD. An increased rate was observed in the case of immobilized enzyme. (2) The soluble high molecular weight derivatives of NAD (dextran-NAD) were immobilized in the collagen membrane with the two dehydrogenases and recycling of dextran-NAD in the membrane was examined. Lactate was produced by the membrane without adding free NAD. The interaction between the high molecular weight NAD derivatives and enzymes are also discussed.     

Enzymatic synthesis of [4R-2H]NAD (P)H and [4S-2H]NAD(P)H and determination of the stereospecificity of 7 alpha- and 12 alpha hydroxysteroid dehydrogenase

The stereospecifically labeled coenzymes [4R-2H]NADH, [4R-2H]NADPH and [4S-2H]NAD(P)H were synthesized enzymatically in high yield and high isotopic purity (greater than or equal to 95%) with 2HCOO2H/formate dehydrogenase, (CH3)2C2HOH/alchol dehydrogenase from Thermoanaerobium brockii and [1-2H]glucose/glucose dehydrogenase, respectively. This set of deuterated coenzymes was used to determine the stereospecificity of the previously unstudied 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli (NAD-dependent) and 12 alpha-hydroxysteroid dehydrogenase from Clostridium group P (NADP-dependent). H-NMR and EI-MS of the nicotinamide moiety after enzymatic oxidation of deuterated NAD(P)H with dehydrocholic acid as substrate showed that both dehydrogenases are B-sterospecific.     

Stereoselective metabolism of the (+)-(S,S)- and (-)-(R,R)-enantiomers of trans-3,4-dihydroxy-3,4-dihydrobenzo[c]-phenanthrene by rat and mouse liver microsomes and by a purified and reconstituted cytochrome P-450 system

Metabolism of (+)-, (-)-, and (+/-)-trans-3,4-dihydroxy-3, 4-dihydrobenzo[c]phenanthrenes by liver microsomes from rats and mice and by a purified monooxygenase system reconstituted with cytochrome P-450c has been examined. Bay-region 3,4-diol 1,2-epoxides are minor metabolites of both enantiomers of the 3,4-dihydrodiol with liver microsomes from 3-methylcholanthrene-treated rats or with the reconstituted system (less than 10% of total metabolites). Microsomes from control and phenobarbital-treated rats and from control mice form higher percentages of these diol epoxides (13-36% of total metabolites). Microsomes from 3-methylcholanthrene-treated rats and cytochrome P-450c in the reconstituted system form exclusively the diol expoxide-1 diastereomer, in which the benzylic hydroxyl group and oxirane oxygen are cis to each other, from the (+)-(3S,4S)-dihydrodiol. The same enzymes selectively form the diol expoxide-2 diastereomer, with its oxirane oxygen and benzylic hydroxyl groups trans to each other, from the (-)-(3R,4R)-dihydrodiol (77% of the total diol epoxides). Liver microsomes from control rats show similar stereoselectivity whereas liver microsomes from phenobarbital-treated rats and from control mice are less stereoselective. Three bis-dihydrodiols and three phenolic dihydrodiols are also formed from the enantiomeric 3,4-dihydrodiols of benzo[c]phenanthrene. A single diastereomer of one of these bis-dihydrodiols with the newly introduced dihydrodiol group at the 7,8-position accounts for 79-88% of the total metabolites of the (-)-(3R,4R)-dihydrodiol formed by liver microsomes from 3-methylcholanthrene-treated rats or by the reconstituted system containing epoxide hydrolase. In contrast, the (+)-(3S,4S)-dihydrodiol is metabolized to two diastereomers of this bis-dihydrodiol, a third bis-dihydrodiol, and two phenolic dihydrodiols.     

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.     

Metabolism of acetoin in mammalian liver slices and extracts. Interconversion with butane-2,3-diol and biacetyl

1. [(14)C]Acetoin was enzymically synthesized from [(14)C]pyruvate with a pyruvate decarboxylase preparation. Its optical activity was [alpha](20) (d)-78 degrees . 2. Large amounts (1000-fold higher than physiological concentrations) of acetoin were incubated with rat liver mince. Acetoin disappeared but very little (14)CO(2) was evolved. A compound accumulated, which was purified and identified as butane-2,3-diol. Chromatography on borate-impregnated paper indicated the presence of both the erythro and threo forms. 3. Liver extracts capable of interconverting biacetyl, acetoin and butane-2,3-diol were obtained. These interconversions were catalysed by two different enzymes: acetoin dehydrogenase (EC 1.1.1.5) and butane-2,3-diol dehydrogenase (EC 1.1.1.4), previously identified in bacteria. Both required NAD(+) or NADP(+) as cofactors and were different from alcohol dehydrogenase. The equilibrium in both cases favoured the more reduced compound. 4. The activity of butane-2,3-diol dehydrogenase was decreased by dialysis against EDTA: the addition of Co(2+), Cu(2+), Zn(2+) and other bivalent metal ions restored activity. 5. Biacetyl reductase was resolved into multiple forms by CM-Sephadex chromatography and electrophoresis.     

Biosynthesis of theobroxide and its related compounds, metabolites of Lasiodiplodia theobromae

Administration of (13)C labeled acetates ([1-(13)C], [2-(13)C] and [1,2-(13)C(2)] to Lasiodiplodia theobromae showed the tetraketide origins of both theobroxide, a potato-tuber inducing substance [1, (1S, 2R, 5S, 6R)-3-methyl-7-oxa-bicyclo[4.1.0]hept-3-en-2,5-diol]) and its carbonyldioxy derivative [2, (1S, 4R, 5S, 6R)-7,9-dioxa-3-methyl-8-oxobicyclo [4.3.0]-2-nonene-4,5-diol]. The incorporation of acetate-derived hydrogen into 1 and 2 was studied using [2-(2)H(3), 2-(13)C]acetate. Three and one deuterium atoms were incorporated at one methyl and epoxy carbons, respectively. The observed loss of deuterium atoms from the methyl group suggests a considerable amount of exchange from the methyl group of [2-(2)H(3), 2-(13)C]acetate during biosynthesis of 1 and 2. Incorporation of [1-(13)C]- and [1,2-(13)C(2)]acetates indicates the carbonyl carbon of the carbonyldioxy derivative is derived from the carboxy carbon of the precursor.     

Combined coenzyme-substrate analogues of various dehydrogenases. Synthesis of (3S)- and (3R)-5-(3-carboxy-3-hydroxypropyl)nicotinamide adenine dinucleotide and their interaction with (S)- and (R)-lactate-specific dehydrogenases.

Two diastereomeric nicotinamide adenine dinucleotide (NAD+) derivatives were synthesized in which the substrates of (S)-and (R)-lactate-specific dehydrogenases are covalently attached via a methylene spacer at position 5 of the nicotinamide ring. The corresponding nicotinamide derivatives were obtained stereospecifically by enzymatic reduction of 5-(2-oxalylethyl)nicotinamide. (3S)-5-(3-Carboxy-3-hydroxypropyl)-NAD+ undergoes and intramolecular hydride transfer in the presence of pig heart lactate dehydrogenase, forming the corresponding coenzyme-substrate analogue composed of pyruvate and NADH. No cross-reaction products resulting from an intermolecular reaction are observed. Two (R)-lactate specific dehydrogenases, however, do not catalyze a similar reaction in either one of the two diastereomers. A possible arrangement of the substrates in the active centers of these enzymes is proposed. 5-Methyl-NAD+ and 5-methyl-NADH are active coenzymes of pig heart lactate dehydrogenase in contrast to reports in the literature. (S)-Lactate binds to this enzyme in the absence of coenzyme, exhibiting a dissociation constant of 11 mM.     

A radiometric technique for measuring nicotinamide adenine dinucleotide in femtomole quantities: methodology and application.

A radiometric technique for measuring as little as 1 × 10^?13 moles (100 femtomoles) of nicotinamide adenine dinucleotide (NAD) is presented. Alcohol dehydrogenase (ADH) (EC 1.1.1.1) is used to oxidize 1-[³H]-ethanol to acetaldehyde in the presence of NAD. The [³H]-NAD so produced is used to reduce pyruvate to [³H]-lactate in the presence of lactic acid dehydrogenase (LDH) (EC 1.1.1.27). In this manner, [³H]-lactate is generated, and NAD is regenerated, permitting the reactions to cycle.After a suitable incubation period, unreacted 1-[³H]-ethanol and acetaldehyde in the reaction mixture are evaporated. Nonvolatile [³H]-lactate is quantitated in a scintillation spectrometer. The applicability of the technique to the measurement of NAD in isolated islets of Langerhans is presented.     

Non-K-region o-quinones as enzyme-generated inactivators of dihydrodiol dehydrogenase

Homogeneous 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from rat liver cytosol catalyzes the NAD(P)+-dependent oxidation of non-K-region trans-dihydrodiols of polycyclic aromatic hydrocarbons, many of which are proximate carcinogens. These reactions proceed with Km values in the millimolar range to yield highly reactive o-quinones that can be trapped as thioether adducts [Smithgall, T. E., Harvey, R. G., & Penning, T. M. (1988) J. Biol. Chem. 263, 1814-1820]. The enzymatically generated o-quinones, e.g., naphthalene-1,2-dione and benzo[a]pyrene-7,8-dione are potent inhibitors of the dehydrogenase, yielding IC50 values of 5.0 and 10.0 microM, respectively. Naphthalene-1,2-dione was found to be an efficient irreversible inhibitor of the enzyme and can inactivate equimolar concentrations of the dehydrogenase, yielding a t 1/2 for the enzyme of 10 s or less. By contrast (+/-)-trans-1,2-dihydroxy-1,2-dihydronaphthalene promotes a slower inactivation of the dehydrogenase, yielding a Kd of 70 microM and a limiting rate constant that corresponds to a t 1/2 at saturation of 23.2 min. Inactivation by this dihydrodiol has an obligatory requirement for NADP+. Examination of the kcat for the oxidation of (+/-)-trans-1,2-dihydroxy-1,2-dihydronaphthalene yields a partition ratio for the dihydrodiol of 200,000, suggesting that alkylation from the parent dihydrodiol is a rare occurrence. Benzo[a]pyrene-7,8-dione, which is the product of the enzymatic oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, also promotes a time- and concentration-dependent inactivation of the dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)