NAD Oxidoreductase Research Articles

Production of OH-radical-type oxidant by lucigenin.

In the presence of NADH- reductases (dihydrolipoamide: NAD oxidoreductase E. C.1.8.1.4 from pig heart or from Clostridium kluyveri; frequently also addressed as "diaphorases") and NADH lucigenin strongly increases ethylene production from a-keto-methylthiobutyrate (KMB) as an indicator for strong oxidants of the OH-radical type. These reactions are further stimulated in the presence of Fe3+ ions. With these NADH-"diaphorases", the structurally similar poison, paraquat, in the absence or presence of Fe3+ has no effect. With ferredoxin-NADP reductase (E. C.1.18.1.2.), however, paraquat reacts quasi identical to lucigenin. Superoxide dismutase, catalase, free radical- or OH-scavengers such as mannitol, propylgallate, DABCO, and desferal inhibit the reaction whereas EDTA (in the presence or absence of added Fe3+) is stimulatory. From these data we conclude that the superoxide--indicator LUC is redox-active after unspecific coupling to several almost ubiquitory NAD(P)H- reductases catalyzing monovalent oxygen reduction. Lucigenin thus should no longer be used as a "specific" superoxide indicator. This report is in agreement with very recent results by Liochev and Fridovich (Arch. Biochem. Biophys. 337, 115 [1997]) and Vasquez-Vivar et al. (FEBS Lett. 403, 127 (1997).

Inosine-5'-monophosphate dehydrogenase is a rate-determining factor for p53-dependent growth regulation.

We have proposed that reduced activity of inosine-5'-monophosphate dehydrogenase (IMPD; IMP:NAD oxidoreductase, EC 1.2.1.14), the rate-limiting enzyme for guanine nucleotide biosynthesis, in response to wild-type p53 expression, is essential for p53-dependent growth suppression. A gene transfer strategy was used to demonstrate that under physiological conditions constitutive IMPD expression prevents p53-dependent growth suppression. In these studies, expression of bax and waf1, genes implicated in p53-dependent growth suppression in response to DNA damage, remains elevated in response to p53. These findings indicate that under physiological conditions IMPD is a rate-determining factor for p53-dependent growth regulation. In addition, they suggest that the impd gene may be epistatic to bax and waf1 in growth suppression. Because of the role of IMPD in the production and balance of GTP and ATP, essential nucleotides for signal transduction, these results suggest that p53 controls cell division signals by regulating purine ribonucleotide metabolism.

Isolation, partial characterization and localization of a dihydrolipoamide dehydrogenase from the cyanobacterium Synechocystis PCC 6803

A dihydrolipoamide dehydrogenase (LPD; dihydrolipoamide:NAD oxidoreductase, EC 1.8.1.4.) activity has been detected in the cyanobacterium Synechocystis PCC 6803. The enzyme was isolated from the membraneous fraction after detergent solubilization and shown to be homogenous on the basis of SDS-PAGE and N-terminal sequencing. The isolated enzyme had a specific activity of 75 U (mg protein) −1 and was shown to be a homodimer with an apparent molecular mass of 104 kDa for the dimer and 55 kDa for the subunits. The enzyme contains 1.75 mol noncovalently bound FAD (mol enzyme) −1 suggesting that each subunit contains 1 mol FAD and that the FAD is fairly tightly associated with the enzyme. N-terminal sequencing gave a contiguous amino acid sequence of 17 residues and showed that the N-terminus of the LPD from Synechocystis PCC 6803 has significant homologies to other LPDs sequenced so far. Immunoblot experiments indicated that the enzyme is mainly present in the membrane fraction, and immunocytochemical investigations gave evidence that the LPD in Synechocystis PCC 6803 is located in the periplasma space between the cytoplasma membrane and the peptidoglycan layer. This is the first report on an extracellular, membrane-bound LPD in a cyanobacterium.

Mycophenolic acid influences Th2-cytokine induced expression on ICAM-1 on human endothelial cells

2-β-D-Ribofuranosyl-4-selenazolecarboxamide (selenazofurin, CI-935), the selenium analog of tiazofurin (CI-909), was 3- to 10-fold more cytotoxic to murine or human tumor cells in vitro than tiazofurin and was also more active against P388 mouse leukemia in vivo. In vitro cytotoxicity could be reversed by guanosine or guanine but not by other purine nucleosides or bases. Three human tumor cell lines selected for selenazofurin or tiazofurin resistance showed crossresistance between selenazofurin and tiazofurin. Treatment with tiazofurin, selenazofurin, or mycophenolic acid decreased guanylate pools and caused an accumulation of IMP in WIL2 human lymphoma cells. The decrease in guanylate pools was accompanied by inhibition of RNA and DNA synthesis. The NAD analogs of tiazofurin and selenazofurin were inhibitors of L1210 IMP dehydrogenase (IMP:NAD oxidoreductase, EC 1.2.1.14), and both showed uncompetitive inhibition with respect to NAD having Kii values of 5.7 × 10−8M and 3.3 × 10−8M respectively.

Ammonium and Glutamate Released by Neurons Are Signals Regulating the Nutritive Function of a Glial Cell

Glial cells transform glucose to a fuel substrate taken up and used by neurons. In the honeybee retina, photoreceptor neurons consume both alanine supplied by glial cells and exogenous proline. Ammonium (NH4+) and glutamate, produced and released in a stimulus-dependent manner by photoreceptor neurons, contribute to the biosynthesis of alanine in glia. Here we report that NH4+ and glutamate are transported into glia and that a transient rise in the intraglial concentration of NH4+ or of glutamate causes a net increase in the level of reduced nicotinamide adenine dinucleotides [NAD(P)H]. Biochemical measurements indicate that this is attributable to activation of glycolysis in glial cells by the direct action of NH4+ and glutamate on at least two enzymatic reactions: those catalyzed by phosphofructokinase (PFK; ATP:D-fructose-6-phosphotransferase, EC2.7.1.11) and glutamate dehydrogenase (GDH; L-glutamate:NAD oxidoreductase, deaminating; EC1.4.1.3). This activation leads to an increase in the production and release of alanine by glia. This signaling, which depends on the rate of conversion of NH4+ and glutamate to alanine and alpha-ketoglutarate, respectively, in the glial cells, raises the novel possibility of a tight regulation of the nutritive function of glia.

Purification and characterization of lactate dehydrogenase from Varanus liver

Abstract Lactate dehydrogenase was purified 21-fold fromliver of Varanus bengalensis using colchicine-sepharose column chromatography. The crudeenzyme showed two isoenzymes (LDH-5 and LDH- 4 )by agarose gel electrophoresis (AGE). The p u r i f i e denzyme showed a single band after SDS-PAGEcorresponding to molecular mass of 35 kDa. Themolecular mass of native enzyme was about 140kDa. The optimum pH for the forward reaction was7.5 while that for the reverse reaction was pH 9.5.The K m values for pyruvate, NADH, lactate andNAD + were 0.17 ± 0.037, 0.02 ± 0.004, 12.4 ± 3.05 and0.38 ± 0.032 mM, respectively. Pre-heating ofenzyme showed that its t 5 0 was 40-50˚C. Oxalateand n-hexanediol were inhibitors for both forwardand reverse reactions. Among divalent ions, Cu + + was shown to be more effective inhibitor for theforward reaction.K e y w o r d s : LDH isoenzyme, liver, properties, purification, Varanus bengalensis Introduction Lactate dehydrogenase ( L -lactate:NAD oxidoreductase,EC 1.1.1.27; LDH) plays an important role in the regu-lation of anaerobic glycolysis through reoxidation ofNADH (Everse and Kaplan, 1973; Javed

Regulation of hydrogen metabolism in Butyribacterium methylotrophicum by substrate and pH

Exogenous H2/CO2 and glucose were consumed simultaneously by Butyribacterium methylotrophicum when grown under glucose-limited conditions. CO2 reduction to acetate was coupled to H2 consumption. The addition of either H2 or CO2 to glucose batch fermentation resulted in an increase in cell density, hydrogenase (H2-consuming and -producing) activities and fatty acid production by B. methylotrophicum as compared to when N2 was the feed gas. Hydrogenase activities appeared to be tightly regulated and were produced at higher rates during the exponential phase when CO2 was the feed gas as compared to H2 or N2. The increase in H2-consuming activity and decrease in H2-producing activity was correlated with an increase in butyrate synthesis. H2-consuming and ferredoxin (Fd)–NAD reductase activities increased while H2-producing and NADH–Fd reductase activities decreased in cells grown at pH 5.5 compared to those at pH 7.0. The molar ratio of butyrate/acetate was shifted from 0.35 at pH 7.0 to 1.22 at pH 5.5. The addition of exogenous H2 did not decrease the butyrate/acetate ratio at pH 7.0 nor at pH 5.5. The results indicated that growth pH values regulated both hydrogenase and Fd–NAD oxidoreductase activities such that, at acid pH, more intermediary electron flow was directed towards butyrate synthesis than H2 production.

Biochemical route and control of butyrate synthesis in Butyribacterium methylotrophicum

Butyribacterium methylotrophicum produced more butyrate when grown on lactate than when grown on glucose, and only acetate was detected during growth on pyruvate. Higher levels of NADH were found in butyrate-producing than in acetate-producing cells. The addition of neutral red, an electron-flow modulator, to cells growing on pyruvate altered the carbon and electron flow from acetate plus H2 synthesis to butyrate synthesis. Enzymatic analysis suggested that pyruvate was produced from glucose via an Embden-Meyerhof-Parnas pathway. Pyruvate was further metabolized to butyryl-CoA via, β-hydroxybutyryl-CoA and butyryl-CoA dehydrogenases. Lactate dehydrogenase, unlike butyryl-CoA dehydrogenase, was inducible and detected only in lactate-grown cells. Both of these dehydrogenases utilized 2,6-dichloroindophenol and other artificial electron acceptors but not NAD(P). Ferredoxin–NAD oxidoreductase levels were highest in lactate and lowest in pyruvate-grown cells. Cells contained both a ferredoxin–neutral-red reductase activity and a neutral-red–NAD reductase activity that coupled electron flow to butyrate synthesis. These results showed that butyrate synthesis by B. methylotrophicum was regulated by the carbon source and was dependent on the cellular NADH/NAD ratios, and the levels and direction of ferredoxin- and NAD-linked oxidoreductases.

Enhanced fermentation of mannitol and release of cytotoxin by Clostridium difficile in alkaline culture media

Clostridium difficile ATCC 43255 fermented less than 10% of the mannitol in a medium at pH 7; however, when the initial pH of the medium was adjusted to 8.5 or 9, about 80% of the mannitol was fermented. Cell extracts of C. difficile phosphorylated mannitol with phosphoenolpyruvate, not ATP, indicating a phosphoenolpyruvate phosphotransferase system transport phosphorylation of mannitol. The phosphorylation product was dehydrogenated by D-mannitol-1-phosphate:NAD oxidoreductase. Growth at an initial pH of 8.5 yielded cytotoxin titers of 10(7) to 10(8) in Trypticase-yeast extract-mannitol medium, wit a titer of 10(8) as early as 13 h.

Molecular cloning, sequencing and expression of a cDNA encoding a human liver NAD-dependent α-glycerol-3-phosphate dehydrogenase

This work reports the primary nucleotide structure and in vitro translation of a cDNA, expressed by a gene mapping on chromosome 12, that encodes a human hepatic α-glycerol-3-phosphate dehydrogenase ( l-glycerol-3-phosphate:NAD oxidoreductase, E.C. 1.1.1.8). The 1413 bp cDNA comprises an ORF of 1050 bp that encodes a 349 amino acid protein of 37.5 kDa. Northern blot analysis of poly(A) + mRNA from human liver showed three transcripts, while from human placenta only two transcripts were detected.

A sensitive determination of uric acid in serum using uricase/catalase/formaldehyde dehydrogenase coupled with formate dehydrogenase

We developed and evaluated an assay for serum uric acid based on the uricase (EC 1.7.3.3)-catalase (EC 1.11.1.6)-formaldehyde dehydrogenase (FADH, EC 1.2.1.46) method coupled with formate dehydrogenase (formate: NAD oxidoreductase, FDH, EC 1.2.1.2). Formate dehydrogenase from Pseudomonas oxalaticus catalyzes the formation of NADH from formate produced by FADH. Owing to the NADH and formate oxidase activity of the FDH itself, the full reaction curve is not linear, but gradually decreases. The formation of NADH is not stoichiometric with formate removal, but is strictly proportional to it. To overcome this decrease of extinction, we added hydroxylamine hydrochloride to the FDH. The sensitivity of the full reaction in the presence of FDH was about 1.8 times that without FDH. Analysis with a Cobas Bio centrifugal analyzer revealed a linearity of up to 3.56 mmol/L. The uricase-catalase-alcohol dehydrogenase method correlated well with the uricase-peroxidase-chromagen method. Our method is more sensitive than other methods.

Effect of polyamines on glutamate dehydrogenase within permeabilized kidney-cortex mitochondria and isolated renal tubules of rabbit

The effect of polyamines on glutamate dehydrogenase { l-glutamate: NAD(P) oxidoreductase (deaminating) [EC 1.4.1.3]} activity has been studied in both permeabilized kidney-cortex mitochondria and isolated renal tubules of rabbit. Spermidine was the most potent inhibitor of glutamate synthesis in permeabilized mitochondria resulting in about 80% decrease of the enzyme activity at 5 mM concentration. Putrescine, α-monofluoromethylputrescine (MFMP) and ( R, R)- δ-methyl- α-acetylenic-putrescine (MAP) were more efficient than spermine. The inhibitory action of polyamines was potentiated by an elevated NADH content in the reaction mixture. Increasing concentrations of either NH 4Cl, KCl or NaCl in the incubation medium resulted in a decrease of polyamine-induced inhibition of the enzyme activity, indicating that monovalent cations can compete with polyamines for the binding site at glutamate dehydrogenase. The inhibitory action of Spermidine on glutamate synthesis was abolished by 2mM ADP or 10 mM l-leucine, allosteric activators of the enzyme, as well as on the addition of either oxalate or sulphate at 20 mM concentrations. Spermidine did not affect glutamate formation when NADH was substituted by NADPH, suggesting an importance of the NADH binding to the inhibitory site of the enzyme for a decrease of reductive amination of 2-oxoglutarate by polyamine. Although Spermidine did not influence glutamate deamination in the presence of NAD +, it stimulated this process by about 70% when NAD + was substituted by NADP + In the presence of ADP the stimulatory effect of polyamine was not significant. The data indicate that in permeabilized rabbit kidney-cortex mitochondria the effect of polyamines on both glutamate formation and glutamate deamination via the reaction catalysed by glutamate dehydrogenase is dependent upon the coenzyme utilized by the enzyme. In the presence of NADH their inhibitory effect on the glutamate formation may be alleviated by allosteric activators of the enzyme, and concentrations of potassium, sodium, sulphate and oxalate. In isolated rabbit renal tubules incubated with 5 mM methionine sulfoximine and aminooxyacetate, in order to inhibit glutamine synthetase and aminotransferases, respectively, 5 mM Spermidine decreased glutamate formation by about 30%, while putrescine and spermine did not significantly diminish the enzyme activity. In the presence of octanoate glutamate formation was reduced by about 30% by naturally occurring polyamines as well as MFMP and MAP, indicating that under these conditions NADH rather than NADPH is utilized as the coenzyme. In view of these data it is possible to suggest that polyamines may be of importance to control glutamate dehydrogenase activity under physiological conditions.

Evidence for a mannitol cycle in Orobanche ramosa and Orobanche crenata

Summary The activities of various enzymes associated with mannitol metabolism in The activities of various enzymes associated with mannitol metabolism in Orobanche ramosa L. and Orobanche crenata Forsk. were measured both in the crude extracts of plant tissue and callus. The detection of D-mannose-6-phosphate ketol isomerase (EC 5.3.1.8), D-mannitol-l-phosphate: NADP oxidoreductase (EC 1.1.1.nn) and D-mannitol-l-phosphate phosphohydrolase (EC 3.1.3.22) activities revealed a similar pathway as postulated by Rumpho et aI. (1983) and Loescher et aI. (1992) for celery and privet, while the appearance of D-mannitol-l-phosphate:NAD oxidoreductase (EC 1.1.1.17) activity in O. callus suggests the possibility of an alternative pathway. To exclude artefacts, the above mentioned enzymes were partially purfied and further characterized. The occurrence of a low affinity D-mannitol: NAD oxidoreductase (EC 1.1.1.67)gives evidence for a mannitol cycle in the two Orobanche species. L. and Orobanche crenata Forsk . were measured both in the crude extracts of plant tissue and callus. The detection of D-mannose-6-phosphate ketol isomerase (EC 5.3.1.8), D-mannitol-1-phosphate: NADP oxidoreductase (EC 1.1.1.nn) and D-mannitol-1-phosphate phosphohydrolase (EC 3.1.3.22) activities revealed a similar pathway as postulated by Rumpho et al. (1983) and Loescher et al. (1992) for celery and privet, while the appearance of D-mannitol-1-phosphate: NAD oxidoreductase (EC 1.1.1.17) activity in O. callus suggests the possibility of an alternative pathway. To exclude artefacts, the above mentioned enzymes were partially purfied and further characterized. The occurrence of a low affinity D-mannitol: NAD oxidoreductase (EC 1.1.1.67) gives evidence for a mannitol cycle in the two Orobanche species.

Valine dehydrogenase from a non-spore-forming bacterium Alcaligenes faecalis: purification and characterization

An NAD-dependent valine dehydrogenase ( L-valine:NAD oxidoreductase, deaminating, EC 1.A.1.-), was found in a non-sporeforming bacterium, Alcaligenes faecalis, and purified about 80-fold to be characterized. The molecular mass of the enzyme was estimated to be about 72 kDa and the enzyme consists of two identical subunits with a molecular mass of 40 kDa and is thermolabile. It loses its activity fully on incubation at 50°C for 5 min. The enzyme catalyzes the reversible deamination of L-valine, which is the preferred substrate, and other branched-chain and straight-chain L-amino acids in the presence of NAD. The pH optima are about 10.8 and 8.8 for the oxidative deamination and reductive amination, respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH was exclusively transferred to the substrate in the reductive amination. The amino-acid composition markedly differs from those of Bacillus sphaericus and Clostridium thermoaceticum leucine dehydrogenases. The enzyme did not react with the antibody of C. thermoaceticum leucine dehydrogenase. Thefore, the enzyme is clearly different from leucine dehydrogenases from spore-forming bacteria, which act on valine, and the first valine dehydrogenase to be characterized in detail.

Purification and Characterization of Clostridium difficile Glutamate Dehydrogenase

Recombinant Clostridium difficile glutamate dehydrogenase (L-glutamate:NAD oxidoreductase, EC 1.4.1.2) was purified 177-fold to electrophoretic homogeneity with a 62% recovery through a four-step procedure involving gel filtration and ion-exchange and dye affinity chromatography. The approximate molecular weights of the native enzyme by gel filtration and subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were consistent with a hexameric structure for the purified enzyme. The enzyme-catalyzed glutamate oxidation was an NAD-dependent sequential process in which NADP could not be substituted as coenzyme. Several dinucleotide analogs of NAD structurally altered in either the pyridine or the purine moiety were observed to function as coenzymes when substituted for NAD. Nicotinamide mononucleotide did not serve as a coenzyme for glutamate oxidation. Product inhibition by NADH was competitive with respect to NAD. In deadend inhibition studies, adenosine diphosphoribose was shown to be an effective coenzyme-competitive inhibitor.

Transformation of N epsilon-CBZ-L-lysine to CBZ-L-oxylysine using L-amino acid oxidase from Providencia alcalifaciens and L-2-hydroxy-isocaproate dehydrogenase from Lactobacillus confusus.

Biotransformations were developed to oxidize N epsilon-carbobenzoxy(CBZ)-L-lysine and to reduce the product keto acid to L-CBZ-oxylysine. Lysyl oxidase (L-lysine: O2 oxidoreductase, EC 1.4.3.14) from Trichoderma viride was relatively specific for L-lysine and had very low activity with N epsilon-substituted derivatives. L-Amino acid oxidase (L-amino acid: O2 oxidoreductase [deaminating], EC 1.4.3.2) from Crotalus adamanteus venom had low activity with L-lysine but high activity with N epsilon-formyl-, t-butyoxycarbonyl(BOC)-, acetyl-, trifluoroacetyl-, or CBZ-L-lysine. L-2-Hydroxyisocaproate dehydrogenase (EC 1.1.1.-) from Lactobacillus confusus catalyzed the reduction by NADH of the keto acids from N epsilon-acetyl-, trifluoroacetyl-, formyl- and CBZ-L-lysine but was inactive with the products from oxidation of L-lysine, L-lysine methyl ester, L-lysine ethyl ester or N epsilon-t-BOC-L-lysine. Providencia alcalifaciens (SC9036, ATCC 13159) was a good microbial substitute for the snake venom oxidase and also provided catalase (H2O2:H2O2 oxidoreductase EC 1.11.1.6). N epsilon-CBZ-L-Lysine was converted to CBZ-L-oxylysine in 95% yield with 98.5% optical purity by oxidation using P. alcalifaciens cells followed by reduction of the keto acid using L-2-hydroxyisocaproate dehydrogenase. NADH was regenerated using formate dehydrogenase (formate: NAD oxidoreductase, EC 1.2.1.2) from Candida boidinii. The Providencia oxidase was localized in the particulate fraction and catalase activity was predominantly in the soluble fraction of sonicated cells. The pH optima and kinetic constants were determined for the reactions.

Alcohol dehydrogenase isoenzymes in rat development: Effect of maternal ethanol consumption

The alcohol dehydrogenase (ADH) isoenzymes (alcohol:NAD oxidoreductase, EC 1.1.1.1) of classes I, III and IV were investigated by activity and starch gel electrophoresis analyses during rat ontogeny. Class I was studied in the liver, class III in the brain and class IV in the stomach and eyes. Classes I and IV exhibited very low activity during the fetal period, reaching 12% and 3%, respectively, of the adult value at birth. Class III was relatively more active in the fetus, with 38% of the adult activity at birth. In the three cases, activity increased after birth and adult values were found around day 20 (classes I and III), day 39 (stomach class IV) and after day 91 (eye class IV). The very low activity of the isoenzymes responsible for ethanol oxidation, i.e. liver class I and stomach class IV, in the fetus demonstrates that metabolism of ethanol during gestation is essentially performed by the maternal tissues. Development of ADH isoenzymes were also studied in the offspring of rats exposed to an alcoholic liquid diet. Activities of liver class I and stomach class IV were severely reduced: they were only 30% and 50%, respectively, of the control values. In contrast, eye class IV activity did not change and brain class III showed a 30% increase. Moreover, the concentration of liver soluble protein exhibited a 1.3–1.5-fold increase with respect to control animals. The effects on activities and liver protein were more pronounced in the adult than in the perinatal period, and they seem irreversible since normal values were not recovered after 6 weeks of feeding with a non-alcoholic diet. The low activities of the alcohol-oxidizing isoenzymes indicate that maternal ethanol consumption results in an impaired ethanol metabolism of the offspring.

Cloning and characterization of a gene from Bacillus stearothermophilus var. non-diastaticus encoding a glycerol dehydrogenase

A 4.1-kb EcoRI fragment which includes the gene ( gldA) encoding a glycerol dehydrogenase (GIDH; EC 1.1.1.6; glycerol:NAD oxidoreductase) from Bacillus stearothermophilus var. non-diastaticus has been cloned by virtue of its ability to restore glycerol utilisation to Escherichia coli glycerol kinase ( glpK) and glycerol-3-phosphate dehydrogenase ( glpD) mutants. Sequencing suggests that the gldA gene is likely to be monocistronic and encodes a protein of 39450 Da. The deduced amino acid composition and sequence of G1DH reveals that the protein is extremely similar to a characterized metal-dependent NAD-dependent G1DH from B. stearothermophilis RS93. The enzyme has limited homology to the iron-activated alcohol dehydrogenase of Zymomonas mobilis and the butanol dehydrogenase of Clostridium acetobutylicum.

Distribution, purification, and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes

Phenylalanine dehydrogenase (L-phenylalanine:NAD oxidoreductase, deaminating; EC 1.4.1.-) was found in various thermophilic actinomycetes. We purified the enzyme to homogeneity from Thermoactinomyces intermedius IFO 14230 by heat treatment and by Red Sepharose 4B, DEAE-Toyopearl, Sepharose CL-4B, and Sephadex G-100 chromatographies with a 13% yield. The relative molecular weight of the native enzyme was estimated to be about 270,000 by gel filtration. The enzyme consists of six subunits identical in molecular weight (41,000) and is highly thermostable: it is not inactivated by incubation at pH 7.2 and 70 degrees C for at least 60 min or in the range of pH 5 to 10.8 at 50 degrees C for 10 min. The enzyme preferably acts on L-phenylalanine and its 2-oxo analog, phenylpyruvate, in the presence of NAD and NADH, respectively. Initial velocity and product inhibition studies showed that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism. The Km values for L-phenylalanine, NAD, phenylpyruvate, NADH, and ammonia were 0.22, 0.078, 0.045, 0.025, and 106 mM, respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH was exclusively transferred to the substrate.

Purification and characterization of diacetyl reductase from chicken liver and Streptococcus lactis and enzymic determination of diacetyl and diketones

Two enzymes which correspond to the Enzyme Commission classification diacetyl reductase (acetoin: NAD oxidoreductase E.C 1.1.1.5) have been isolated and purified from chicken liver and Streptococcus lactis subspecies diacetylactis 18-16. Both enzymes reduced vicinal diketones and uncharged α-dicarbonyl compounds using NADH as cofactor; the liver enzyme also accepted NADPH as cofactor. The bacterial enzyme accepted acetoin as a substrate whereas the liver enzyme did not. The liver enzyme was used to quantify aqueous diacetyl or diketone solutions by enzymic reaction with reduced cofactor. The quantity of reduced cofactor remaining after reaction was inversely proportional to the original concentration of diacetyl or diketones. The relationship between absorbance and concentration was linear up to 50 mg dm −3 diacetyl, with a lower limit of sensitivity of 1 mg dm −3. Greater sensitivity (linearity up to 1.5 mg dm −3, lower limit of 0.05 mg dm −3) could be obtained by coupling the reaction to an enzymic cycling system.