Glutamate Synthase: Properties of the Reduced Nicotinamide Adenine Dinucleotide-Dependent Enzyme from Saccharomyces cerevisiae

A reduced nicotinamide adenine dinucleotide (NADH)-dependent glutamate synthase has been detected and partially purified from crude extracts of Saccharomyces cerevisiae. The enzyme is specific for NADH, glutamine, and alpha-ketoglutarate (K(m) values of 2.6 muM, 1.0 mM, and 140 muM, respectively) and has a pH optimum between 7.1 and 7.7. The stoichiometry of the reaction has been determined as 2 mol of glutamate synthesized per mol of glutamine consumed. Glutamate synthase can be distinguished from either of the glutamate dehydrogenases of yeast on the basis of its substrate requirements and behavior during agarose gel and ion exchange chromatography. Variations in the specific activity of glutamate synthase, which occur in response to changes in the growth medium, are similar in character to those observed with the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase.     

Glutamate dehydrogenase from Mycoplasma laidlawii

Mycoplasma laidlawii possesses a single glutamate dehydrogenase (GDH) with dual coenzyme specificity [specificity for nicotinamide adenine dinucleotide (H) and nicotinamide adenine dinucleotide phosphate (H)]. A purification procedure is reported which results in an enzyme preparation with a specific activity of 79.5 units/mg and which displays only one significant protein band after gel electrophoresis. This one band was determined, by activity staining, to have all of the GDH nucleotide specificities. The molecular weight of the enzyme is 250,000 +/- 10%, and it has a subunit size of about 48,000. The enzyme exhibits measurable activity with aspartate and pyruvate but is inactive with eight other possible substrates. Purine nucleotides do not affect the activity. The K(m) for reduced nicotinamide adenine dinucleotide was 1.8 x 10(-4)m. The optimal substrate concentrations and pH optimum for each of the respective GDH activities are also reported.     

Overall kinetic mechanism of saccharopine dehydrogenase from Saccharomyces cerevisiae

Kinetic data have been measured for the histidine-tagged saccharopine dehydrogenase from Saccharomyces cerevisiae, suggesting the ordered addition of nicotinamide adenine dinucleotide (NAD) followed by saccharopine in the physiologic reaction direction. In the opposite direction, the reduced nicotinamide adenine dinucleotide (NADH) adds to the enzyme first, while there is no preference for the order of binding of alpha-ketoglutarate (alpha-Kg) and lysine. In the direction of saccharopine formation, data also suggest that, at high concentrations, lysine inhibits the reaction by binding to free enzyme. In addition, uncompetitive substrate inhibition by alpha-Kg and double inhibition by NAD and alpha-Kg suggest the existence of an abortive E:NAD:alpha-Kg complex. Product inhibition by saccharopine is uncompetitive versus NADH, suggesting a practical irreversibility of the reaction at pH 7.0 in agreement with the overall K(eq). Saccharopine is noncompetitive versus lysine or alpha-Kg, suggesting the existence of both E:NADH:saccharopine and E:NAD:saccharopine complexes. NAD is competitive versus NADH, and noncompetitive versus lysine and alpha-Kg, indicating the combination of the dinucleotides with free enzyme. Dead-end inhibition studies are also consistent with the random addition of alpha-Kg and lysine. Leucine and oxalylglycine serve as lysine and alpha-Kg dead-end analogues, respectively, and are uncompetitive against NADH and noncompetitive against alpha-Kg and lysine, respectively. Oxaloacetate (OAA), pyruvate, and glutarate behave as dead-end analogues of lysine, which suggests that the lysine-binding site has a higher affinity for keto acid analogues than does the alpha-Kg site or that dicarboxylic acids have more than one binding mode on the enzyme. In addition, OAA and glutarate also bind to free enzyme as does lysine at high concentrations. Glutarate gives S-parabolic noncompetitive inhibition versus NADH, indicating the formation of a E:(glutarate)2 complex as a result of occupying both the lysine- and alpha-Kg-binding sites. Pyruvate, a slow alternative keto acid substrate, exhibits competitive inhibition versus both lysine and alpha-Kg, suggesting the combination to the E:NADH:alpha-Kg and E:NADH:lysine enzyme forms. The equilibrium constant for the reaction has been measured at pH 7.0 as 3.9 x 10(-7) M by monitoring the change in NADH upon the addition of the enzyme. The Haldane relationship is in very good agreement with the directly measured value.     

Partial purification and some properties of delta1-pyrroline-5-carboxylate reductase from Escherichia coli.

delta1-Pyrroline-5-carboxylate (PCA) reductase [L-proline:NAD(P)+5-oxidoreductase, EC 1.5.1.2] has been purified over 200-fold from Escherichia coli K-12. It has a molecular weight of approximately 320,000. PCA reductase mediates the pyridine nucleotide-linked reduction of PCA to proline but not the reverse reaction (even at high substrate concentrations). The partially purified preparation is free of competing pyridine nucleotide oxidase, PCA dehydrogenase, and proline oxidase activities. The Michaelis constant (Km) values for the substrate, PCA, with reduced nicotinamide adenine dinucleotide phosphate (NADPH) or NADH as cofactor are 0.15 and 0.14 mM, respectively. The Km values determined for NADPH and NADH are 0.03 and 0.23 mM, respectively. Although either NADPH or NADH can function as cofactor, the activity observed with NADPH is severalfold greater. PCA reductase is not repressed by growth in the presence of proline, but it is inhibited by the reaction end products, proline and NADP.     

Properties of the nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase from Blastocladiella emersonii.

The nicotinamide adenine dinucleotide phosphate (NADP)-specific isocitrate dehydrogenase from Blastocladiella emersonii was purified. The enzyme was very unstable. Satisfactory stability was obtained in the presence of 0.2% ovalbumin. The enzyme had a molecular weight of about 100,000. It did not exhibit homotropic cooperativity for any of it substrates and was not affected by the allosteric modifiers citrate and adenosine monophosphate, diphosphate, and tri-phosphate. The substrate saturation studies showed both intercept and slope effects in Lineweaver-Burk plots. The Km values for isocitrate and NADP were found to be 20 and 10 muM, respectively. The product inhibition pattern was compatible with a random sequential reaction mechanism. The enzyme catalyzed the oxidative decarboxylation of isocitrate about six times better than the reductive carboxylation of alpha-ketoglutarate. The enzyme was inhibited by glyoxylate plus oxalacetate. Assays conducted in the presence of low Mg2+ concentrations exhibited a lag. This lag could be abolished by the addition of reduced NADP to the assay mixture.     

Nitrite reductase of Escherichia coli specific for reduced nicotinamide adenine dinucleotide

Kemp, John D. (University of California, Los Angeles), and Daniel E. Atkinson. Nitrite reductase of Escherichia coli specific for reduced nicotinamide adenine dinucleotide. J. Bacteriol. 92:628-634. 1966.-A nitrite reductase specific for reduced nicotinamide adenine dinucleotide (NADH(2)) appears to be responsible for in vivo nitrite reduction by Escherichia coli strain Bn. In extracts, the reduction product is ammonium, and the ratio of NADH(2) oxidized to nitrite reduced or to ammonium produced is 3. The Michaelis constant for nitrite is 10 mum. The enzyme is induced by nitrite, and the ability of intact cells to reduce nitrite parallels the level of NADH(2)-specific nitrite reductase activity demonstrable in cell-free preparations. Crude extracts of strain Bn will also reduce hydroxylamine, but not nitrate or sulfite, at the expense of NADH(2). Kinetic observations indicate that hydroxylamine and nitrite may both be reduced at the same active site. The high apparent Michaelis constant for hydroxylamine (1.5 mm), however, seems to exclude hydroxylamine as an intermediate in nitrite reduction. In vitro activity is enhanced by preincubation with nitrite, and decreased by preincubation with NADH(2).     

Nucleoside Diphosphate-Sugar 4-Epimerases: II. Uridine Diphosphate Arabinose 4-Epimerase of Wheat Germ 1

Uridine diphosphate (UDP)-arabinose 4-epimerase (EC 5.1.3.5) has been purified at least 20-fold from wheat germ by MnCl(2) treatment, (NH(4))(2)SO(4) fractionation, dialysis, and Sephadex and diethylaminoethyl cellulose column chromatography. The enzyme has no action on UDP-d-glucose, UDP-d-glucuronic acid, or TDP-d-glucose. The pH optimum is 8.0. Km values are 1.5 mM for UDP-d-xylose and 0.5 mm for UDP-l-arabinose. The equilibrium constant, K, for the reaction UDP-l-arabinose left arrow over right arrow UDP-d-xylose is 1.25. The enzyme is neither activated by nicotinamide adenine dinucleotide nor inhibited by reduced nicotinamide adenine dinucleotide. It is completely inhibited by p-chloromercuri-phenylsulfonate; the inhibition is reversed by cysteine.     

Purification and properties of the glutathione reductase of Chromatium vinosum.

The Chromatium vinosum glutathione reductase [NAD(P)H: glutathione disulfide oxidoreductase, EC 1.6.4.2] was purified to apparent homogeneity. The enzyme was found to require reduced nicotinamide adenine dinucleotide (NADH) as a reductant and to be specific for oxidized glutathione (GSSG). The polypeptide molecular weight in sodium dodecyl sulfate was found to be 52,000. Incubation of enzyme with NADH in the absence of GSSG resulted in a significant loss in activity. The enzyme was stimulated by phosphate and sulfate ion, but was inhibited by chloride ion, heavy metals, and sulfhydryl reagents. Adenylate nucleotides were inhibitory, and the data suggested that they were acting as competitive inhibitors of flavin adenine dinucleotide (FAD). The Km values of 7 X 10-3 for GSSG and 6 X 10-5 M for NADH were the highest reported of any previously investigated glutathione reductase. The order of addition of components markedly affected the response of the enzyme to FAD. A requirement for FAD (Km 5.2 X 10-7 M) was seen if the enzyme was incubated with NADH prior to GSSG addition, whereas no FAD was required if the order was reversed.     

Purification and properties of glutamate synthase from Thiobacillus thioparus.

Glutamate synthase was purified about 250-fold from Thiobacillus thioparus and was characterized. The molecular weight was estimated as 280,000 g/mol. The enzyme showed absorption maxima at 280, 380, and 450 nm and was inhibited by Atebrin, suggesting that T. thioparus glutamate synthase is a flavoprotein. The enzyme activity was also inhibited by iron chelators and thiolbinding agents. The enzyme was specific for reduced nicotinamide adenine dinucleotide phosphate (NADPH) and alpha-ketoglutarate, but L-glutamine was partially replaced by ammonia as the amino donor. The Km values of glutamate synthase for NADPH, alpha-ketoglutarate, and glutamine were 3.0 muM, 50 muM, and 1.1 mM, respectively. The enzyme had a pH optimum between 7.3 and 7.8. Glutamate synthase from T. thioparus was relatively insensitive to feedback inhibition by single amino acids but was sensitive to the combined effects of several amino acids. Enzymes involved in glutamate synthesis in T. thioparus were studied. Glutamine synthetase and glutamate synthase, as well as two glutamate dehydrogenases (NADH and NADPH dependent), were present in this organism. This levels of glutamate synthase and glutamate dehydrogenase were similar in T. thioparus grown on 0.7 or 7.0 mM ammonium sulfate. The sum of the activities of both glutamate dehydrogenases was only 1/25 of that of glutamate synthase under the assay conditions. It was concluded that the glutamine pathway is important for ammonia assimilation in this autotrophic bacterium.     

Inhibition by N'-nitrosonornicotine of the catalytic activity of glutamate dehydrogenase in alpha-ketoglutarate amination

The effect of N'-nitrosonornicotine (NNN), one of the tobacco-specific nitrosamines, on the catalytic activity of glutamate dehydrogenase (GLDH) in the alpha-ketoglutarate amination, using reduced nicotinamide adenine dinucleotide as coenzyme, was studied by a chronoamperometric method. The maximum reaction rate of the enzyme-catalyzed reaction and the Michaelis-Menten constant, or the apparent Michaelis-Menten constant, were determined in the absence and presence of NNN. NNN remarkably inhibited the bio-catalysis activity of GLDH, and was a reversible competitive inhibitior with K(i), estimated as 199 micromol l(-1) at 25 degrees C and pH 8.0.     

Effects of diethylstilbestrol, 2,2'-dithiodipyridine, and chloral hydrate on the esterase activity of sheep liver cytoplasmic aldehyde dehydrogenase

The binding of diethylstilbestrol (DES) to aldehyde dehydrogenase (ALDH) has a very similar effect on the dehydrogenase activity of the enzyme as has modification of the enzyme by 2,2'-dithiodipyridine [Kitson, T.M. (1982) Biochem. J. 207, 81-89]. The latter modification may occur at the site of the esterase activity of the enzyme [Kitson, T.M. (1985) Biochem. J. 228, 765-767]. This suggests that DES might be a competitive inhibitor of the esterase reaction. However, in the absence of oxidized nicotinamide adenine dinucleotide (NAD+) or reduced nicotinamide adenine dinucleotide (NADH), and at low concentrations of substrate (4-nitrophenyl acetate, PNPA), DES is a potent partial noncompetitive inhibitor. It is concluded therefore that DES binds at a site different from the esterase active site and that the enzyme-DES complex retains some ability to act as an esterase. High concentrations of PNPA appear to displace DES from its binding site. In the presence of NAD+, DES is a weaker inhibitor, and in the presence of NADH, DES has very little effect. Esterase activity is enhanced by NADH when PNPA concentrations are high but is inhibited when they are low. The rate of reaction of ALDH with 2,2'-dithiodipyridine is only slightly reduced by DES, suggesting that the site at which thiol modifiers react and the DES binding site are different. When ALDH is modified by 2,2'-dithiodipyridine, it has reduced esterase activity, which declines further as the modified enzyme loses its 2-thiopyridyl label. In the presence of NAD+, chloral hydrate is a simple competitive inhibitor of the esterase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new ternary complex between horse liver alcohol dehydrogenase, NAD+, and acetone

Acetone was found to form a dead-end ternary complex with horse liver alcohol dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD⁺) when the reactants were incubated for a long time at relatively high concentrations. The complex formation was demonstrated by measuring the increase in absorbance at 320 nm, the quenching of protein fluorescence, and the loss of enzyme activity. Since acetone is a substrate of liver alcohol dehydrogenase, and the presence of acetaldehyde or pyrazole prevents acetone from forming the dead-end complex with liver alcohol dehydrogenase and NAD⁺, the acetone molecule in the complex may be bound to the substrate binding site of liver alcohol dehydrogenase. The dissociation of the complex was demonstrated by prolonged dialysis or by addition of reduced nicotinamide adenine dinucleotide (NADH) and iso-butyramide. A modified nicotinamide adenine dinucleotide was obtained as a main product from the dead-end complex after dissociation of the complex or denaturation of the apoenzyme. The modified nicotinamide adenine dinucleotide was found to exhibit an absorption spectrum similar to that of NADH; however, it was not oxidizable by liver alcohol dehydrogenase in the presence of acetaldehyde and exhibited no fluorescence.     

Flavensomycin, an inhibitor of enzyme reactions involving hydrogen transfer

The antifungal antibiotic flavensomycin inhibited the oxidation of amino acids and of glucose by Penicillium oxalicum. The compound inhibited l-amino acid oxidase (EC 1.4.3.2) activity for l-leucine and l-phenylalanine, and also d-amino acid oxidase (EC 1.4.3.3) in the oxidation for dl-alanine. The addition of flavin adenine dinucleotide, which is a cofactor for this enzyme, antagonized the action of the antibiotic. Glucose oxidase (EC 1.1.3.4) was also inhibited. The antibiotic inhibited the reduced nicotinamide adenine dinucleotide (NADH(2)) cytochrome c reductase (EC 1.6.2.1) as well as the much slower nonenzymatic reduction of this cytochrome by the nucleotide. Reduced cytochrome c was also oxidized nonenzymatically by flavensomycin. The antibiotic completely inhibited the action of rabbit muscle lactic dehydrogenase (EC 1.1.1.27) in promoting the reduction of pyruvate by NADH(2) but only slightly affected the reverse reaction. Alcohol dehydrogenase (EC 1.1.1.1) was also similarly inhibited. Flavensomycin prevented the reduction of nicotinamide adenine dinucleotide phosphate by isocitrate in the presence of isocitrate dehydrogenase (EC 1.1.1.42). The hexokinase (EC 2.7.1.1)-catalyzed phosphorylation of glucose, in which the adenosine triphosphate acts as a phosphate donor, was only slightly affected. Flavensomycin also inhibited the action of yeast lactate dehydrogenase (EC 1.1.2.3) on the reduction of cytochrome c. High concentrations of cytochrome c were antagonistic to this reaction. The results point to an interference with enzymatically controlled hydrogen or electron transfer as the mechanism of the antifungal activity of flavensomycin.     

Alpha-glycerophosphate oxidase in Streptococcus faecium F 24

alpha-Glycerophosphate oxidase, in a strain of Streptococcus faecium, was found to be adaptive to aerated conditions of growth. The enzyme was purified and found to mediate electron transfer from alpha-glycerophosphate to O(2), with the production of stoichiometric concentrations of H(2)O(2) and dihydroxyacetone phosphate. The enzyme is an anionic flavoprotein, with flavine adenine dinucleotide as the apparent prosthetic group. By manometric methods, a K(m) of 6 x 10(-3)m, with reference to substrate concentration, was obtained. An active reduced nicotinamide adenine dinucleotide diaphorase was closely associated with this enzyme in chromatographic mobility on ECTEOLA-cellulose. The purified alpha-glycerophosphate oxidase was not inhibited by KCN, azide, or sulfhydryl reagents, nor was it stimulated by alpha-lipoate, yeast extract, or other supplements.     

Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum: Purification and Properties

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase in Clostridium thermoaceticum used, in addition to its natural electron acceptor, methyl and benzyl viologen. The enzyme was purified to a specific activity of 34 (micromoles per minute per milligram of protein) with NADP as electron acceptor. Disc gel electrophoresis of the purified enzyme yielded two major and two minor protein bands, and during centrifugation in sucrose gradients two components of apparent molecular weights of 270,000 and 320,000 were obtained, both having formate dehydrogenase activity. The enzyme preparation catalyzed the reduction of riboflavine 5'-phosphate flavine adenine dinucleotide and methyl viologen by using reduced NADP as a source of electrons. It also had reduced NADP oxidase activity. The enzyme was strongly inhibited by cyanide and ethylenediaminetetraacetic acid. It was also inhibited by hypophosphite, an inhibition that was reversed by formate. Sulfite inhibited the activity with NADP but not with methyl viologen as acceptor. The apparent K(m) at 55 C and pH 7.5 for formate was 2.27 x 10(-4) M with NADP and 0.83 x 10(-4) with methyl viologen as acceptor. The apparent K(m) for NADP was 1.09 x 10(-4) M and for methyl viologen was 2.35 x 10(-3) M. NADP showed substrate inhibition at 5 x 10(-3) M and higher concentrations. With NADP as electron acceptor, the enzyme had a broad pH optimum between 7 and 9.5. The apparent temperature optimum was 85 C. In the absence of substrates, the enzyme was stable at 70 C but was rapidly inactivated at temperatures above 73 C. The enzyme was very sensitive to oxygen but was stabilized by thiol-iron complexes and formate.     

A salt-inhibited cytochrome c reductase obtained from the moderately halophilic bacterium, Micrococcus halodenitrificans

A membrane-bound cytochrome c reductase from Micrococcus halodenitrificans was inhibited by NaCl at concentrations as low as 0.05 m. The inhibition was competitive with respect to cytochrome c and noncompetitive with respect to reduced nicotinamide adenine dinucleotide. Thus the effect of NaCl was to increase the apparent K(m) of the enzyme for cytochrome c and not to inhibit the intrinsic activity of the enzyme.     

Ferrisiderophore reductase activity in Bacillus megaterium.

The release of iron from ferrisiderophores (microbial ferric-chelating iron transport cofactors) by cell-free extracts of Bacillus megaterium was demonstrated. Reductive transfer of iron from ferrisiderophores to the ferrous-chelating agent ferrozine was measured spectrophotometrically. This ferrisiderophore reductase activity (reduced nicotinamide adenine dinucleotide phosphate:ferrisiderophore oxidoreductase) was associated primarily with the cell soluble rather than particulate (membrane) fraction. Ferrisiderophore reductase was inhibited by oxygen and required the addition of a reductant (reduced nicotinamide adenine dinucleotide phosphate was most effective) for maximal activity. The activity was destroyed by both heat and protease treatments and was inhibited by iodoacetamide treatment. Ferrisiderophore reductase activity for several microbial ferrisiderophores was measured; highest activity was displayed for ferrischizokinen, the ferrisiderophore produced by this organism. The Km and Vmax values of the reductase for ferrischizokinen were 2.5 x 10(-4) M and 35.7 nmol/min per mg of the ferrisiderophore reductase reaction. Preliminary fractionation of the cell soluble material by gel filtration chromatography resulted in the demonstration of ferrisiderophore reductase activity in three peaks of different molecular weight. Ferrisiderophore reductase probably mediates entrance of iron into cellular metabolism.     

Mutagen Stability of Alkylation-Sensitive Mutants of Bacillus subtilis

The phosphotransacetylase of Veillonella alcalescens catalyzes a reversible reaction with Michaelis-Menten kinetics for all substrates. The rate of the reverse reaction (the synthesis of acetyl coenzyme A from acetyl phosphate) was 6.5 times greater than the rate of the forward reaction (the synthesis of acetyl phosphate from acetyl coenzyme A). The apparent K(m) values determined for the forward reaction were 8.6 x 10(-6)m for acetyl coenzyme A and 9.3 x 10(-3)m for phosphate. In the reverse reaction, the K(m) values were 3.3 x 10(-4)m for coenzyme A and 5.9 x 10(-4)m for acetyl phosphate. The results of an analysis of the inhibition by end products in the forward and reverse directions were compatible with a random bi- bi- mechanism. The enzyme was inhibited by adenosine triphosphate and adenosine diphosphate but was not affected by reduced nicotinamide adenine dinucleotide or pyruvate. The inhibition by adenosine triphosphate was noncompetitive with respect to acetyl phosphate and competitive with respect to coenzyme A. MgCl(2) reversed the inhibition by adenosine triphosphate or adenosine diphosphate. The role of Mg(2+) and adenylates in the regulation of phosphotranscetylase activity is discussed.     

Kinetic and mechanistic properties of biotin sulfoxide reductase

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase catalyzes the reduction of d-biotin d-sulfoxide (BSO) to biotin. Initial rate studies of the homogeneous recombinant enzyme, expressed in Escherichia coli, have demonstrated that the purified protein utilizes NADPH as a facile electron donor in the absence of any additional auxiliary proteins. We have previously shown [Pollock, V. V., and Barber, M. J. (1997) J. Biol. Chem. 272, 3355-3362] that, at pH 8 and in the presence of saturating concentrations of BSO, the enzyme exhibits, a marked preference for NADPH (k(cat,app) = 500 s(-1), K(m,app) = 269 microM, and k(cat,app)/K(m,app) = 1.86 x 10(6) M(-1) s(-1)) compared to NADH (k(cat,app) = 47 s(-1), K(m,app) = 394 microM, and k(cat,app)/K(m,app) = 1.19 x 10(5) M(-1) s(-1)). Production of biotin using NADPH as the electron donor was confirmed by both the disk biological assay and by reversed-phase HPLC analysis of the reaction products. The purified enzyme also utilized ferricyanide as an artificial electron acceptor, which effectively suppressed biotin sulfoxide reduction and biotin formation. Analysis of the enzyme isolated from tungsten-grown cells yielded decreased reduced methyl viologen:BSO reductase, NADPH:BSO reductase, and NADPH:FR activities, confirming that Mo is required for all activities. Kinetic analyses of substrate inhibition profiles revealed that the enzyme followed a Ping Pong Bi-Bi mechanism with both NADPH and BSO exhibiting double competitive substrate inhibition. Replots of the 1/v-axes intercepts of the parallel asymptotes obtained at several low concentrations of fixed substrate yielded a K(m) for BSO of 714 and 65 microM for NADPH. In contrast, utilizing NADH as an electron donor, the replots yielded a K(m) for BSO of 132 microM and 1.25 mM for NADH. Slope replots of data obtained at high concentrations of BSO yielded a K(i) for BSO of 6.10 mM and 900 microM for NADPH. Kinetic isotope studies utilizing stereospecifically deuterated NADPD indicated that BSO reductase uses specifically the 4R-hydrogen of the nicotinamide ring. Cyanide inhibited NADPH:BSO and NADPH:FR activities in a reversible manner while diethylpyrocarbonate treatment resulted in complete irreversible inactivation of the enzyme concomitant with molybdenum cofactor release, indicating that histidine residues are involved in cofactor-binding.     

Presence of Escherichia coli of a deaminase and a reductase involved in biosynthesis of riboflavin.

Two enzymes have been partially purified from extracts of Escherchia coli B which together catalyze the conversion of the product of the action of GTP cyclohydrolase II, 2,5-diamino-6-oxy-4-(5'-phosphoribosylamine)pyrimidine, to 5-amino-2,6-dioxy-4-(5'-phosphoribitylamine)pyrimidine. These two compounds are currently thought to be intermediates in the biosynthesis of riboflavin. The enzymatic conversion occurs in two steps. The product of the action of GTP cyclohydrolase II first undergoes hydrolytic deamination at carbon 2 of the ring, followed by reduction of the ribosylamino group to a ribitylamino group. The enzyme which catalyzes the first step, herein called the "deaminase," has been purified 200-fold. The activity was assayed by detecting the conversion of the product of the reaction catalyzed by GTP cyclohydrolase II to a compound which reacts with butanedione to form 6,7-dimethyllumazine. The enzyme has a molecular weight of approximately 80,000 and a pH optimum of 9.1. The dephosphorylated form of the substrate is not deaminated in the presence of the enzyme. The assay for the enzyme which catalyzes the second step, referred to here as the "reductase," involves the detection of the conversion of the product of the deaminase-catalyzed reaction to a compound which, after treatment with alkaline phosphatase, reacts with butanedione to form 6,7-dimethyl-8-ribityllumazine. The reductase has a molecular weight of approximately 40,000 and a pH optimum of 7.5. Like the deaminase, the reductase does not act on the dephosphorylated form of its substrate. Reduced nicotinamide adenine dinucleotide phosphate is required as a cofactor; reduced nicotinamide adenine dinucleotide can be used about 30% as well as the phosphate form. The activity of neither enzyme is inhibited by riboflavin, FMN, or flavine adenine dinucleotide.