Properties and synthesis regulation of NAD(P)H dehydrogenases from Rhodobacter capsulatus

Three NAD(P)H dehydrogenases were found and purified from a soluble fraction of cells of the purple non-sulfur bacterium Rhodobacter capsulatus, strain B10. Molecular mass of NAD(P)H, NADPH and NADH dehydrogenases are 67 000 (4 · 18 000), 35 000 and 39 000, and the isoelectric points are 4.6, 4.3 and 4.5, respectively. NAD(P)H dehydrogenase is characterized by a higher sensitivity to quinacrine, NADPH dehydrogenase by its sensitivity to p-chloromercuribenzoate and NADH dehydrogenase by its sensitivity to sodium arsenite. In contrast to the other two enzymes, NAD(P)H dehydrogenase is capable of oxidizing NADPH as well as NADH, but the ratio of their oxidation rates depends on the pH. All NAD(P)H dehydrogenases reacted with ferricyanide, 2,6-dichlorophenolindophenol, benzoquinone and naphthoquinone, but did not exhibit transhydrogenase, reductase or oxidase activity. Moreover, NADH dehydrogenase was also capable of reducing FAD and FMN. NAD(P)H and NADH dehydrogenases possessed cytochrome-c reductase activity, which was stimulated by menadione and ubiquinone Q₁. The activity of NAD(P)H and NADH dehydrogenases depended on culture-growth conditions. The activity of NAD(P)H dehydrogenase from cells grown under chemoheterotrophic aerobic conditions was the lowest and it increased notably under photoheterotrophic anaerobic conditions upon lactate or malate growth limitation. The activity of NADH dehydrogenase was higher from the cells grown under photoheterotrophic anaerobic conditions upon nitrate growth limitation and under chemoheterotrophic aerobic conditions. NADPH dehydrogenase synthesis dependence on R. capsulatus growth conditions was insignificant.     

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae)

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q₀, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K _m-values for both pyridine nucleotides were in the molar range (K _m[NADH]=9.8 M, K _m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP⁺ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP⁺ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme Q₀-2,3-dimethoxy-5-methyl-1,4-benzoquinone - FNR ferredoxin: NADP⁺ reductase - MD menadione - MV methylviologen - NDH NAD(P)H dehydrogenase - PQ plastoquinone - PQ₁₀ decylplastoquinone - SDH succinate dehydrogenase - UQ₁₀ decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone)     

Effects of the administration of anticoagulants on the activity of the enzyme-reduced NAD(P)H dehydrogenase in rat livers, hepatomas and precarcinomatous rat liver lesions

The anticoagulants dicumarol, warfarin and diphenadione, are in vitro inhibitors of the enzyme reduced NAD(P)H dehydrogenase of rat liver. These chemicals were administered by intragastric gavage to determine whether the same inhibitory effects could be observed in Sprague-Dawley male rats. Doses of 2 and 10 mg/100 g body weight were used. Our results indicate that only dicumarol inhibited the enzyme, whereas warfarin did not produce a significant effect, and diphenadione at large doses produced an increase in the activity of the enzyme. Dicumarol was further tested to see if it would alter the activity of the enzyme in hyperplastic nodules and liver hepatomas. A similar inhibitory effect was found. The three strains of rats tested in this work have different levels of reduced NAD(P)H dehydrogenase activity. Thus, our results indicate that dicumarol is the only anticoagulant that inhibits in vivo the reduced NAD(P)H dehydrogenase of rat liver and liver neoplasms.     

Dual coenzyme activities of high-Km aldehyde dehydrogenase from rat liver mitochondria

Various kinetic approaches were carried out to investigate kinetic attributes for the dual coenzyme activities of mitochondrial aldehyde dehydrogenase from rat liver. The enzyme catalyses NAD(+)- and NADP(+)-dependent oxidations of ethanal by an ordered bi-bi mechanism with NAD(P)+ as the first reactant bound and NAD(P)H as the last product released. The two coenzymes presumably interact with the kinetically identical site. NAD+ forms the dynamic binary complex with the enzyme, while the enzyme-NAD(P)H complex formation is associated with conformation change(s). A stopped-flow burst of NAD(P)H formation, followed by a slower steady-state turnover, suggests that either the deacylation or the release of NAD(P)H is rate limiting. Although NADP+ is reduced by a faster burst rate, NAD+ is slightly favored as the coenzyme by virtue of its marginally faster turnover rate.     

Stereo-specificity for pro-(R) hydrogen of NAD(P)H during enzyme-catalyzed hydride transfer to CL-20

A dehydrogenase from Clostridium sp. EDB2 and a diaphorase from Clostridium kluyveri were reacted with CL-20 to gain insights into the enzyme-catalyzed hydride transfer to CL-20, and the enzyme's stereo-specificity for either pro-R or pro-S hydrogens of NAD(P)H. Both enzymes biotransformed CL-20 at rates of 18.5 and 24nmol/h/mg protein, using NADH and NADPH as hydride-source, respectively, to produce a N-denitrohydrogenated product with a molecular weight of 393Da. In enzyme kinetics studies using reduced deuterated pyridine nucleotides, we found a kinetic deuterium isotopic effect of 2-fold on CL-20 biotransformation rate using dehydrogenase enzyme against (R)NADD as a hydride-source compared to either (S)NADD or NADH. Whereas, in case of diaphorase, the kinetic deuterium isotopic effect of about 1.5-fold was observed on CL-20 biotransformation rate using (R)NADPD as hydride-source. In a comparative study with LC-MS, using deuterated and non-deuterated NAD(P)H, we found a positive mass-shift of 1Da in the N-denitrohydrogenated product suggesting the involvement of a deuteride (D(-)) transfer from NAD(P)D. The present study thus revealed that both dehydrogenase and diaphorase enzymes from the two Clostridium species catalyzed a hydride transfer to CL-20 and showed stereo-specificity for pro-R hydrogen of NAD(P)H.     

Reduction of Fe(III) ions complexed to physiological ligands by lipoyl dehydrogenase and other flavoenzymes in vitro: implications for an enzymatic reduction of Fe(III) ions of the labile iron pool

Enzymatic reduction of physiological Fe(III) complexes of the "labile iron pool" has not been studied so far. By use of spectrophotometric assays based on the oxidation of NAD(P)H and formation of [Fe(II) (1,10-phenanthroline)3]2+ as well as by utilizing electron paramagnetic resonance spectrometry, it was demonstrated that the NAD(P)H-dependent flavoenzyme lipoyl dehydrogenase (diaphorase, EC 1.8.1.4) effectively catalyzes the one-electron reduction of Fe(III) complexes of citrate, ATP, and ADP at the expense of the co-enzymes NAD(P)H. Deactivated or inhibited lipoyl dehydrogenase did not reduce the Fe(III) complexes. Likewise, in the absence of NAD(P)H or in the presence of NAD(P)+, Fe(III) reduction could not be detected. The fact that reduction also occurred in the absence of molecular oxygen as well as in the presence of superoxide dismutase proved that the Fe(III) reduction was directly linked to the enzymatic activity of lipoyl dehydrogenase and not mediated by O2. Kinetic studies revealed different affinities of lipoyl dehydrogenase for the reduction of the low molecular weight Fe(III) complexes in the relative order Fe(III)-citrate > Fe(III)-ATP > Fe(III)-ADP (half-maximal velocities at 346-485 microm). These Fe(III) complexes were enzymatically reduced also by other flavoenzymes, namely glutathione reductase (EC 1.6.4.2), cytochrome c reductase (EC 1.6.99.3), and cytochrome P450 reductase (EC 1.6.2.4) with somewhat lower efficacy. The present data suggest a (patho)physiological role for lipoyl dehydrogenase and other flavoenzymes in intracellular iron metabolism.     

Membrane-bound sugar alcohol dehydrogenase in acetic acid bacteria catalyzes L-ribulose formation and NAD-dependent ribitol dehydrogenase is independent of the oxidative fermentation

To identify the enzyme responsible for pentitol oxidation by acetic acid bacteria, two different ribitol oxidizing enzymes, one in the cytosolic fraction of NAD(P)-dependent and the other in the membrane fraction of NAD(P)-independent enzymes, were examined with respect to oxidative fermentation. The cytoplasmic NAD-dependent ribitol dehydrogenase (EC 1.1.1.56) was crystallized from Gluconobacter suboxydans IFO 12528 and found to be an enzyme having 100 kDa of molecular mass and 5 s as the sedimentation constant, composed of four identical subunits of 25 kDa. The enzyme catalyzed a shuttle reversible oxidoreduction between ribitol and D-ribulose in the presence of NAD and NADH, respectively. Xylitol and L-arabitol were well oxidized by the enzyme with reaction rates comparable to ribitol oxidation. D-Ribulose, L-ribulose, and L-xylulose were well reduced by the enzyme in the presence of NADH as cosubstrates. The optimum pH of pentitol oxidation was found at alkaline pH such as 9.5-10.5 and ketopentose reduction was found at pH 6.0. NAD-Dependent ribitol dehydrogenase seemed to be specific to oxidoreduction between pentitols and ketopentoses and D-sorbitol and D-mannitol were not oxidized by this enzyme. However, no D-ribulose accumulation was observed outside the cells during the growth of the organism on ribitol. L-Ribulose was accumulated in the culture medium instead, as the direct oxidation product catalyzed by a membrane-bound NAD(P)-independent ribitol dehydrogenase. Thus, the physiological role of NAD-dependent ribitol dehydrogenase was accounted to catalyze ribitol oxidation to D-ribulose in cytoplasm, taking D-ribulose to the pentose phosphate pathway after being phosphorylated. L-Ribulose outside the cells would be incorporated into the cytoplasm in several ways when need for carbon and energy sources made it necessary to use L-ribulose for their survival. From a series of simple experiments, membrane-bound sugar alcohol dehydrogenase was concluded to be the enzyme responsible for L-ribulose production in oxidative fermentation by acetic acid bacteria.     

Characterization of a C21 neutral steroid hormone transforming enzyme, 21-dehydroxylase, in crude cell extracts of Eubacterium lentum.

A strain of the obligate anaerobe, Eubacterium lentum, isolated from human feces, catalyzes the 21-dehydroxylation of 11-deoxycorticosterone to progesterone. A quantitative radiochromatographic assay was developed to measure 21-dehydroxylase activity in cell extracts. Maximum enzyme activity in cell extracts required both a reduced pyridine nucleotide and an oxidized flavin coenzyme. However, photochemically reduced flavin (FMNH2) could replace the requirement for NAD(P)H plus oxidized flavin. NAD(P)H : flavin (either FMN or FAD) oxidoreductase activity was detected spectrophotometrically in cell extracts assayed under anaerobic conditions. 21-Dehydroxylase was active from pH 5.4 to 8.5 with an apparent optimum between 6.4 and 6.8 using mixtures of NADH plus FMN as coenzymes. The substrate concentration at half-maximal reaction velocity was 8.0 microM and a specific acitivity of 5.8 nmol [3H]progesterone formed . h-1 . mg-1 protein was determined using [3th]deoxycorticosterone as substrate. Atabrine, rotenone, acriflavin, and 2,4-dinitrophenol (all at 1 mM) inhibited 21-dehydroxylase activity in cell extracts by 25, 24, 35 and 84%, respectively. These results suggest that 21-dehydrogenase may be coupled to a NAD(P)H : flavin oxidoreductase system in E. lentum.     

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.     

A specific radiochemical assay for pyrroline-5-carboxylate dehydrogenase

Previous studies of pyrroline-5-carboxylate dehydrogenase have been conducted using a spectrophotometric method to monitor substrate-dependent NAD(P)H production. For the assay of the mammalian enzyme, the spectrophotometric assay was found to be unacceptable for kinetic studies as the production of NAD(P)H was nonlinear with time and protein concentration. An assay which measures radiolabeled glutamate production by this enzyme in the presence of NAD+ from radiolabeled pyrroline-5-carboxylate has been developed. Separation of substrate from product is achieved by column chromatography using Dowex 50 cation-exchange resin. The product isolated by this procedure was identified as glutamate. This new assay is linear with time and protein concentration and gives reproducible results. The assay is not influenced by competing enzyme activities, such as glutamate dehydrogenase, in a liver homogenate so that quantitative conversion of pyrroline-5-carboxylate to glutamate is observed.     

Bioluminescent enzyme immunoassay for progesterone using monoclonal antibodies and glucose-6-phosphate dehydrogenase labels

Bacterial luciferase, NAD(P): FMN oxidoreductase and anti-mouse immunoglobulin were co-immobilized on Sepharose 4B. This reagent together with a progesterone glucose-6-phosphate dehydrogenase conjugate and various anti-progesterone monoclonal antibodies was used to develop a non-separation bioluminescent immunoassay for progesterone. This monoclonal antibody based assay was sensitive and reliable and using the tracer progesterone-11-acetate-glucose-6-phosphate dehydrogenase, the majority of the monoclonal antibodies give a better sensitivity with this enzymatic tracer than that obtained with an iodinated tracer. In a second assay design progesterone-glutathione was co-immobilized with bacterial luciferase and NAD(P): FMN oxidoreductase on Sepharose 4B and three monoclonal antibodies were labelled with glucose-6-phosphate dehydrogenase. With aqueous progester-one standards, this assay gave comparable sensitivity to the bioluminescent enzyme immunoassay using the second antibody immunoadsorbant and to an RIA but was unsuitable for plasma samples.     

Succinate Dehydrogenase and Other Respiratory Pathways in Thylakoid Membranes of Synechocystis sp. Strain PCC 6803: Capacity Comparisons and Physiological Function

Respiration in cyanobacterial thylakoid membranes is interwoven with photosynthetic processes. We have constructed a range of mutants that are impaired in several combinations of respiratory and photosynthetic electron transport complexes and have examined the relative effects on the redox state of the plastoquinone (PQ) pool by using a quinone electrode. Succinate dehydrogenase has a major effect on the PQ redox poise, as mutants lacking this enzyme showed a much more oxidized PQ pool. Mutants lacking type I and II NAD(P)H dehydrogenases also had more oxidized PQ pools. However, in the mutant lacking type I NADPH dehydrogenase, succinate was essentially absent and effective respiratory electron donation to the PQ pool could be established after addition of 1 mM succinate. Therefore, lack of the type I NADPH dehydrogenase had an indirect effect on the PQ pool redox state. The electron donation capacity of succinate dehydrogenase was found to be an order of magnitude larger than that of type I and II NAD(P)H dehydrogenases. The reason for the oxidized PQ pool upon inactivation of type II NADH dehydrogenase may be related to the facts that the NAD pool in the cell is much smaller than that of NADP and that the NAD pool is fully reduced in the mutant without type II NADH dehydrogenase, thus causing regulatory inhibition. The results indicate that succinate dehydrogenase is the main respiratory electron transfer pathway into the PQ pool and that type I and II NAD(P)H dehydrogenases regulate the reduction level of NADP and NAD, which, in turn, affects respiratory electron flow through succinate dehydrogenase.     

Covalent fixation of NAD+ to dehydrogenases and properties of the modified enzymes

Starting from 6-chloropurine riboside and NAD+, different reactive analogues of NAD+ have been obtained by introducing diazoniumaryl or aromatic imidoester groups via flexible spacers into the nonfunctional adenine moiety of the coenzyme. The analogues react with different amino-acid residues of dehydrogenases and form stable amidine or azobridges, respectively. After the formation of a ternary complex by the coenzyme, the enzyme and a pseudosubstrate, the reactive spacer is anchored in the vicinity of the active site. Thus, the coenzyme remains covalently attached to the protein even after decomposition of the complex. On addition of substrates the covalently bound coenzyme is converted to the dihydro-form. In enzymatic tests the modified dehydrogenases show 80-90% of the specific activity of the native enzymes, but they need remarkably higher concentrations of free NAD+ to achieve these values. The dihydro-coenzymes can be reoxidized by oxidizing agents like phenazine methosulfate or by a second enzyme system. Various systems for coenzyme regeneration were investigated; the modified enzymes were lactate dehydrogenase from pig heart and alcohol dehydrogenase from horse liver; the auxiliary enzymes were alcohol dehydrogenase from yeast and liver, lactate dehydrogenase from pig heart, glutamate dehydrogenase and alanine dehydrogenase. Lactate dehydrogenase from heart muscle is inhibited by pyruvate. With alanine dehydrogenase as the auxiliary enzyme, the coenzyme is regenerated and the reaction product, pyruvate, is removed. This system succeeds to convert lactate quantitatively to L-alanine. The thermostability of the binary enzyme systems indicates an interaction of covalently bound coenzymes with both dehydrogenases; both binding sites seem to compete for the coenzyme. The comparison of dehydrogenases with different degrees of modifications shows that product formation mainly depends on the amount of incorporated coenzyme.     

Preparation of a NAD(H)-polymer matrix showing coenzyme function of the bound pyridine nucleotide

To a Sepharose gel the pyridine nucleotide NAD(H) has been bound using dicyclohexyl carbodiimide. In order to improve the steric availability of the nucleotide for added soluble enzymes such as dehydrogenases, a spacer molecule, ε-amino caproic acid, was inserted between the carbohydrate matrix and the nucleotide. The obtained preparation contained 56 μmoles NAD⁺/g dry polymer. The obtained matrix-bound NAD(H) was accepted as coenzyme by added lactate dehydrogenase. These preparations were still active after storage for several weeks at 4° C and could be used repeatedly without loss of activity. This represents the first necessary step taken in the preparation of compact closed systems consisting of “enzyme–coenzyme–coenzyme-regenerating enzyme” bound to individual polymer beads; such systems eliminate the need for continuous coenzyme addition.     

耐热醋酸棱状芽孢杆菌中具有高的二硫键还原活性组分的提取和特性研究

用ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ层析柱从Ｃ．ｔｈｅｒｍｏａｃｅｔｉｃｕｍ细胞提取物中分离出的两个具有相近分子量（７００）的活性组分,在ＭＶ＋存在下,对二硫键具有高的催化还原活性．这两组分参与的催化还原反应不以ＮＡＤ（Ｐ）Ｈ为电子供体．在实验条件下,对二硫键的催化还原活性顺序为：ＧＳＳＧ＞硫辛酰胺＞胱氨酸＞硫辛酸．活性组分具有较高的反应稳定性和热稳定性．两组分在２６０、３５４和５０５（４６５）ｎｍ处具有特征吸收峰．     

Regeneration of lipophilic antioxidants by NAD(P)H:quinone oxidoreductase 1

Summary.  The aim of this work was to study the activity of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2) in the regeneration of lipophilic antioxidants, alpha-tocopherol, and reduced-coenzyme Q analogs. First, we tested whether or not two isoforms of the NAD(P)H:(quinone acceptor) oxidoreductase 1 designated as “hydrophilic” and “hydrophobic” (H. J. Prochaska and P. Talalay, Journal of Biological Chemistry 261: 1372–1378, 1986) show differential enzyme activities towards hydrophilic or hydrophobic ubiquinone homologs. By chromatography on phenyl Sepharose, we purified the two isoforms from pig liver cytosol and measured their reduction of several ubiquinone homologs of different side chain length. We also studied by electron paramagnetic resonance the effect of NAD(P)H:(quinone acceptor) oxidoreductase 1 on steady-state levels of chromanoxyl radicals generated by linoleic acid and lipooxygenase and confirmed the enzyme's ability to protect alpha-tocopherol against oxidation induced with H₂O₂-Fe²⁺. Our results demonstrated that the different hydrophobicities of the isoforms do not reflect different reactivities towards ubiquinones of different side chain length. In addition, electron paramagnetic resonance studies showed that in systems containing the reductase plus NADH, levels of chromanoxyl radicals were dramatically reduced. Morever, in the presence of oxidants, alpha-tocopherol was preserved by NAD(P)H:(quinone acceptor) oxidoreductase 1, supporting our hypothesis that regeneration of alpha-tocopherol may be one of the physiologic functions of this enzyme. Received May 20, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Edificio Severo Ochoa, Campus de Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.     

Regulation of ox liver glutamate dehydrogenase activity by coenzymes

The effects of coenzymes NAD(P) and NAD(P)H on the kinetics of the ox liver glutamate dehydrogenase reaction have been studied. The oxidized coenzymes were shown to activate alpha-ketoglutarate amination at inhibiting concentrations of NADH and NADPH. The reduced coenzymes, NADH and NADPH, inhibit glutamate deamination with both NAD and NADP as coenzymes. The data obtained are discussed in terms of literature data on the mechanisms of the coenzyme effects on the glutamate dehydrogenase activity and are inconsistent with the theory of direct ligand--ligand interactions. It was shown that the peculiarities of the glutamate dehydrogenase kinetics can easily be interpreted in the light of the two state models.     

Kinetic and stereochemical characterization of hamster liver 3 alpha-hydroxysteroid dehydrogenase and 3 alpha(17 beta)-hydroxysteroid dehydrogenase

The kinetic mechanism of NADP(+)-dependent 3 alpha-hydroxysteroid dehydrogenase and NAD(+)-dependent 3 alpha(17 beta)-hydroxysteroid dehydrogenase, purified from hamster liver cytosol, was studied in both directions. For 3 alpha-hydroxysteroid dehydrogenase, the initial velocity and product inhibition studies indicated that the enzyme reaction sequence is ordered with NADP+ binding to the free enzyme and NADPH being the last product to be released. Inhibition patterns by Cibacron blue and hexestrol, and binding studies of coenzyme and substrate are also consistent with an ordered bi bi mechanism. For 3 alpha(17 beta)-hydroxysteroid dehydrogenase, the steady-state kinetic measurements and substrate binding studies suggest a random binding pattern of the substrates and an ordered release of product; NADH is released last. However, the two enzymes transferred the pro-R-hydrogen atom of NAD(P)H to the carbonyl substrate.     

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.