S-formylglutathione: The substrate for formate dehydrogenase in methanol-utilizing yeasts

Formaldehyde dehydrogenase and formate dehydrogenase were purified 45- and 16-fold, respectively, from Hansenula polymorpha grown on methanol. Formaldehyde dehydrogenase was strictly dependent on NAD and glutathione for activity. The K _mvalues of the enzyme were found to be 0.18 mM for glutathione, 0.21 mM for formaldehyde and 0.15 mM for NAD. The enzyme catalyzed the glutathine-dependent oxidation of formaldehyde to S-formylglutathione. The reaction was shown to be reversible: at pH 8.0 a K _mof 1 mM for S-formylglutathione was estimated for the reduction of the thiol ester with NADH. The enzyme did not catalyze the reduction of formate with NADH. The NAD-dependent formate dehydrogenase of H. polymorpha showed a low affinity for formate (K _mof 40 mM) but a relatively high affinity for S-formylglutathione (K _mof 1.1 mM). The K _mvalues of formate dehydrogenase in cell-free extracts of methanol-grown Candida boidinii and Pichia pinus for S-formylglutathione were also an order of magnitude lower than those for formate. It is concluded that S-formylglutathione rather than free formate is an intermediate in the oxidation of methanol by yeasts.     

Purification and properties of formate dehydrogenase from Moraxella sp. strain C-1

NAD+-dependent formate dehydrogenase was screened in various bacterial strains. Facultative methanol-utilizing bacteria isolated from soil samples, acclimated to a medium containing methanol and formate at pH 9.5, were classified as members of the genus Moraxella. From a crude extract of Moraxella sp. strain C-1, formate dehydrogenase was purified to homogeneity, as judged by disc gel electrophoresis. The enzyme has an isoelectric point of 3.9 and a molecular weight of approximately 98,000. The enzyme is composed of two identical subunits with molecular weights of about 48,000. The apparent Km values for sodium formate and NAD+ were calculated to be 13 mM and 0.068 mM, respectively.     

Properties of glutamate dehydrogenase purified from Bacteroides fragilis

The dual pyridine nucleotide-specific glutamate dehydrogenase [EC 1.4.1.3] was purified 37-fold from Bacteroides fragilis by ammonium sulfate fractionation, DEAE-Sephadex A-25 chromatography twice, and gel filtration on Sephacryl S-300. The enzyme had a molecular weight of approximately 300,000, and polymeric forms (molecular weights of 590,000 and 920,000) were observed in small amounts on polyacrylamide gel disc electrophoresis. The molecular weight of the subunit was 48,000. The isoelectric point of the enzyme was pH 5.1. This glutamate dehydrogenase utilized NAD(P)H and NAD(P)+ as coenzymes and showed maximal activities at pH 8.0 and 7.4 for the amination with NADPH and with NADH, respectively, and at pH 9.5 and 9.0 for the deamination with NADP+ and NAD+, respectively. The amination activity with NADPH was about 5-fold higher than that with NADH. The Lineweaver-Burk plot for ammonia showed two straight lines in the NADPH-dependent reactions. The values of Km for substrates were: 1.7 and 5.1 mM for ammonium chloride, 0.14 mM for 2-oxoglutarate, 0.013 mM for NADPH, 2.4 mM for L-glutamate, and 0.019 mM for NADP+ in NADP-linked reactions, and 4.9 mM for ammonium chloride, 7.1 mM for 2-oxoglutarate, 0.2 mM for NADH, 7.3 mM for L-glutamate, and 3.0 mM for NAD+ in NAD-linked reactions. 2-Oxoglutarate and L-glutamate caused substrate inhibition in the NADPH- and NADP+-dependent reactions, respectively, to some extent. NAD+- and NADH-dependent activities were inhibited by 50% by 0.1 M NaCl. Adenine nucleotides and dicarboxylic acids did not show remarkable effects on the enzyme activities.     

A single cyclohexadienyl dehydrogenase specifies the prephenate dehydrogenase and arogenate dehydrogenase components of the dual pathways to L-tyrosine in Pseudomonas aeruginosa

Dual biosynthetic pathways diverge from prephenate to L-tyrosine in Pseudomonas aeruginosa, with 4-hydroxyphenylpyruvate and L-arogenate being the unique intermediates of these pathways. Prephenate dehydrogenase and arogenate dehydrogenase activities could not be separated throughout fractionation steps yielding a purification of more than 200-fold, and the ratio of activities was constant throughout purification. Thus, the enzyme is a cyclohexadienyl dehydrogenase. The native enzyme has a molecular weight of 150,000 and is a hexamer made up of identical 25,500 subunits. The enzyme is specific for NAD+ as an electron acceptor, and identical Km values of 0.25 mM were obtained for NAD+, regardless of whether activity was assayed as prephenate dehydrogenase or as arogenate dehydrogenase. Km values of 0.07 mM and 0.17 mM were calculated for prephenate and L-arogenate, respectively. Inhibition by L-tyrosine was noncompetitive with respect to NAD+, but was strictly competitive with respect to either prephenate or L-arogenate. With cyclohexadiene as variable substrate, similar Ki values for L-tyrosine of 0.06 mM (prephenate) and 0.05 mM (L-arogenate) were obtained. With NAD+ as the variable substrate, similar Ki values for L-tyrosine of 0.26 mM (prephenate) and 0.28 mM (L-arogenate), respectively, were calculated. This is the first characterization of a purified, monofunctional cyclohexadienyl dehydrogenase.     

Purification and properties of an NAD(P)+-linked formaldehyde dehydrogenase from Methylococcus capsulatus (Bath).

Crude soluble extracts of Methylococcus capsulatus strain Bath, grown on methane, were found to contain NAD(P)+-linked formaldehyde dehydrogenase activity. Activity in the extract was lost on dialysis against phosphate buffer, but could be restored by supplementing with inactive, heat-treated extract (70 degrees C for 12 min). The non-dialysable, heat-sensitive component was isolated and purified, and has a molecular weight of about 115000. Sodium dodecyl sulphate gel electrophoresis of the protein suggested there were two equal subunits with molecular weights of 57000. The heat-stable fraction, which was necessary for activity of the heat-sensitive protein, was trypsin-sensitive and presumed to be a low molecular weight protein or peptide. A number of thiol compounds and other common cofactors could not replace the component present in the heat-treated soluble extract. The purified formaldehyde dehydrogenase oxidized three other aldehydes with the following Km values: 0.68 mM (formaldehyde); 0.075 mM (glyoxal); 7.0 mM (glycolaldehyde); and 2.0 mM (DL-glyceraldehyde). NAD+ or NADP+ was required for activity, with Km values of 0.063 and 0.155 mM respectively, and could not be replaced by any of the artificial electron acceptors tested. The enzyme was heat-stable at 45 degrees C for at least 10 min and had temperature and pH optima of 45 degrees C and pH 7.2 respectively. A number of metal-binding agents and substrate analogues were not inhibitory. Thiol reagents gave varying degrees of inhibition, the most potent being p-hydroxymercuribenzoate which at 1 mM gave 100% inhibition. The importance of possessing an NAD(P)+-linked formaldehyde dehydrogenase, with respect to M. capsulatus, is discussed.     

L-Threonine dehydrogenase from goat liver. Feedback inhibition by methylglyoxal

L-Threonine dehydrogenase, which forms aminoacetone from L-threonine and NAD, has been extensively purified from goat liver. A feedback inhibition of this enzyme has been observed with methylglyoxal. Kinetic data and other experiments indicate that methylglyoxal acts at a site other than the active site of the enzyme. The enzyme contains a single subunit of Mr 89,000. The apparent Km values of the enzyme for L-threonine and NAD were found to be 5.5 and 1 mM, respectively.     

Purification and characterization of formaldehyde dehydrogenase from rat liver cytosol.

Formaldehyde dehydrogenase was purified to electrophoretic and column chromatographic homogeneity from rat liver cytosolic fraction by a procedure which includes ammonium sulfate precipitation, DEAE-cellulose-, hydroxyapatite-, Mono Q-chromatography, and gel filtration. Its molecular mass was estimated to be 41 kDa by gel filtration and SDS-PAGE, suggesting that it is a monomer. It utilized neither methylglyoxal nor aldehydes except formaldehyde as a substrate. It has been reported that liver class III alcohol dehydrogenase and formaldehyde dehydrogenase are the same enzyme and oxidize formaldehyde and long chain primary alcohols. However, the enzyme examined here did not use n-octanoi as a substrate. The Km values for formaldehyde and NAD+ were 5.09 and 2.34 microM at 25 degrees C, respectively. The amino acid sequences of 10 peptides obtained from the purified enzyme after digestion with either V8 protease or lysyl endopeptidase were determined. From these results, the enzyme was proved to be different from the previously described mammalian formaldehyde dehydrogenase and is the first true formaldehyde dehydrogenase to be isolated from a mammalian source.     

Purification and properties of beta-hydroxybutyrate dehydrogenase from Mycobacterium phlei ATCC354.

beta-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) was purified 145-fold from Mycobacterium phlei ATCC354 by ammonium sulphate fractionation and DEAE-cellulose chromatography. The pH optima for oxidation and reduction reactions were 8.4 and 6.8 respectively. The purified enzyme was specific for NAD, NADH, acetoacetate and D(-)-beta-hydroxybutyrate. Km values for DL-beta-hydroxybutyrate and NAD were 7.4 mM and 0.66 mM respectively. The enzyme was inactivated by mercurial thiol inhibitors and by heat, but could be protected by NADH, Ca2+ and partially by Mn2+. The enzyme did not require metal ions and was insensitive to EDTA, glutathione, dithiothreitol, beta-mercaptoethanol and cysteine.     

The artificial evolution of an enzyme by random mutagenesis: the development of formaldehyde dehydrogenase

A unique variant of glutathione independent formaldehyde dehydrogenase of Pseudomonas putida was obtained by random mutagenesis using the PCR-reaction. This YM042 mutant, S318G, was a cold-adapted formaldehyde dehyrogenase. The activity at 29 degrees C of the variant was 1.7-fold higher than that of the wild type. The K(m) values of the mutant at 37 degrees C were 0.40 mM for NAD(+) and 2.5 mM for formaldehyde, while those of the wild-type were 0.18 mM for NAD(+) and 2.1 mM for formaldehyde. The catalytic efficiency for formaldehyde was about 1.5-fold greater in the mutant than in the wild-type enzyme. The optimum pHs and temperatures of the mutant and the wild-type enzyme were 7.5, and 8.0 and 37 degrees C, and 47 degrees C, respectively. The thermal stability of the mutant was lower than that of the wild type.     

NAD-linked formate dehydrogenase from methanol-grown Pichia pastoris NRRL-Y-7556

An NAD-linked formate dehydrogenase (EC 1.2.1.2.) from methanol-grown Pichia pastoris NRRL Y-7556 has been purified. The purification procedure involved ammonium sulfate fractionation, hollow-fiber H1P10 filtration, ion-exchange chromatography, and gel filtration. Both dithiothreitol (10 mm) and glycerol (10%) were required for stability of the enzyme during purification. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis and by sedimentation pattern in an ultracentrifuge. The enzyme has a molecular weight of 94,000 and consists of two subunits of identical molecular weight. Formate dehydrogenase catalyzes specifically the oxidation of formate. No other compounds tested can replace NAD as the electron acceptor. The Michaelis constants were 0.14 mm for NAD and 16 mm for formate (pH 7.0, 25 °C). Optimum pH and temperature for formate dehydrogenase activity were around 6.5–7.5 and 20–25 °C, respectively. Amino acid composition of the enzyme was also studied. Antisera prepared against the purified enzyme from P. pastoris NRRL Y-7556 form precipitin bands with isofunctional enzymes from different strains of methanol-grown yeasts, but not bacteria, on immunodiffusion plates. Immunoglobulin fraction prepared against the enzyme from yeast strain Y-7556 inhibits the catalytic activity of the isofunctional enzymes from different strains of methanol-grown yeasts.     

Purification and characterization of 4-N-trimethylamino-1-butanol dehydrogenase of Pseudomonas sp. 13CM

A new enzyme, NAD+-dependent 4-N-trimethylamino-1-butanol dehydrogenase from Pseudomonas sp. 13CM, was purified 526-fold to apparent homogeneity in 5 chromatographic steps. The enzyme had a molecular mass of 45 kDa and appeared to be a monomer enzyme. The isoeletric point was found to be 4.8. The optimum temperature was 50 degrees C, and the optimum pHs for the oxidation and reduction reactions were 9.5 and 6.0 respectively. The purified enzyme was further characterized with respect to substrate specificity, kinetic parameters, and amino acid terminal sequence. The Km values for trimethylamino-1-butanol and NAD+ were 0.54 mM and 0.22 mM respectively. In the reduction reaction, the apparent Km values for trimethylaminobutylaldehyde and NADH were 0.67 mM and 0.04 mM, respectively. The enzyme was inhibited by SH reagents, chelating reagents, and heavy metal ions. The N-terminal 12 amino acid residues were sequenced.     

Alanine dehydrogenase from Streptomyces fradiae. Purification and properties

Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210,000 or 205,000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51,000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L-alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH4+ and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.     

Purification and some properties of NAD(P)H dehydrogenase from Saccharomyces cerevisiae: Contribution to glycolytic methylglyoxal pathway

NAD(P)H dehydrogenase was purified approximately 480-fold from Saccharomyces cerevisiae with 6.5% activity yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 40,000–44,000 by gel filtration on Sephadex G-150 column chromatography and SDS-polyacrylamide gel electrophoresis. The K_m values for NADPH and NADH were 7.3 μM and 0.1 mM, respectively. The activity of the enzyme increased approximately 4-fold with Cu²⁺. FAD, FMN and cytochrome c were not effective as electron acceptors, although Fe(CN)₆³⁻ was slightly effective. NADH generated by the reaction of lactaldehyde dehydrogenase in the glycolytic methylglyoxal pathway will be reoxidized by NAD(P)H dehydrogenase. NAD(P)H dehydrogenase thus may contribute to the reduction/oxidation system in the glycolytic methylglyoxal pathway to maintain the flux of methylglyoxal to lactic acid via lactaldehyde.     

The first archaeal L-aspartate dehydrogenase from the hyperthermophile Archaeoglobus fulgidus: gene cloning and enzymological characterization

A gene encoding an L-aspartate dehydrogenase (EC 1.4.1.21) homologue was identified in the anaerobic hyperthermophilic archaeon Archaeoglobus fulgidus. After expression in Escherichia coli, the gene product was purified to homogeneity, yielding a homodimeric protein with a molecular mass of about 48 kDa. Characterization revealed the enzyme to be a highly thermostable L-aspartate dehydrogenase, showing little loss of activity following incubation for 1 h at up to 80 degrees C. The optimum temperature for L-aspartate dehydrogenation was about 80 degrees C. The enzyme specifically utilized L-aspartate as the electron donor, while either NAD or NADP could serve as the electron acceptor. The Km values for L-aspartate were 0.19 and 4.3 mM when NAD or NADP, respectively, served as the electron acceptor. The Km values for NAD and NADP were 0.11 and 0.32 mM, respectively. For reductive amination, the Km values for oxaloacetate, NADH and ammonia were 1.2, 0.014 and 167 mM, respectively. The enzyme showed pro-R (A-type) stereospecificity for hydrogen transfer from the C4 position of the nicotinamide moiety of NADH. This is the first report of an archaeal L-aspartate dehydrogenase. Within the archaeal domain, homologues of this enzyme occurred in many Methanogenic species, but not in Thermococcales or Sulfolobales species.     

Substrate specificity of bovine liver formaldehyde dehydrogenase

Formaldehyde dehydrogenases isolated from several different biological sources have been reported to catalyze the NAD+-dependent oxidative acylation of glutathione by methylglyoxal to form S-pyruvylglutathione, suggesting the involvement of this enzyme in the metabolism of methylglyoxal. However, formaldehyde dehydrogenase from bovine liver is found not to use methylglyoxal or related alpha-ketoaldehydes as substrates. Using methylglyoxal with the enzyme under conditions favoring the forward reaction did not result in the formation of S-pyruvylglutathione. Using independently synthesized S-pyruvylglutathione with the enzyme under conditions favoring the reverse reaction did not result in the production of methylglyoxal. In addition, methylglyoxal and several related alpha-ketoaldehydes did not exhibit detectable activity with formaldehyde dehydrogenase partially purified from human liver, contrary to a previous report. Some, if not all, past reports that methylglyoxal serves as a substrate for the dehydrogenase may be due to the demonstrated presence of contaminating formaldehyde in some commercially available preparations of methylglyoxal. In a related study, S-hydroxymethylglutathione, formed by pre-equilibrium addition of formaldehyde to glutathione, is concluded to be direct substrate for the dehydrogenase. This follows from the observation that the catalytic turnover number of the enzyme in the forward direction exceeds by a factor of approximately 20 the first order rate constant for decomposition of S-hydroxymethylglutathione to glutathione and formaldehyde (k = 5.03 +/- 0.30 min-1, pH 8, 25 degrees C).     

Different forms of rat liver aldehyde dehydrogenase and their subcellular distribution.

1. The properties and distribution of the NAD-linked unspecific aldehyde dehydrogenase activity (aldehyde: NAD+ oxidoreductase EC 1.2.1.3) has been studied in isolated cytoplasmic, mitochondrial and microsomal fractions of rat liver. The various types of aldehyde dehydrogenase were separated by ion exchange chromatography and isoelectric focusing. 2. The cytoplasmic fraction contained 10-15, the mitochondrial fraction 45-50 and the microsomal fraction 35-40% of the total aldehyde dehydrogenase activity, when assayed with 6.0 mM propionaldehyde as substrate. 3. The cytoplasmic fraction contained two separable unspecific aldehyde dehydrogenases, one with high Km for aldehydes (in the millimolar range) and the other with low Km for aldehydes (in the micromolar range). The latter can, however, be due to leakage from mitochondria. The high-Km enzyme fraction contained also all D-glucuronolactone dehydrogenase activity of the cytoplasmic fraction. The specific formaldehyde and betaine aldehyde dehydrogenases present in the cytoplasmic fraction could be separated from the unspecific activities. 4. In the mitochondrial fraction there was one enzyme with a low Km for aldehydes and another with high Km for aldehydes, which was different from the cytoplasmic enzyme. 5. The microsomal aldehyde dehydrogenase had a high Km for aldehydes and had similar properties as the mitochondrial high-Km enzyme. Both enzymes have very little activity with formaldehyde and glycolaldehyde in contrast to the other aldehyde dehydrogenases. They are apparently membranebound.     

Steady-state kinetics of formaldehyde dehydrogenase and formate dehydrogenase from a methanol-utilizing yeast, Candida boidinii.

Initial velocity studies and product inhibition studies were conducted for the forward and reverse reactions of formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC 1.2.1.1) isolated from a methanol-utilizing yeast Candida boidinii. The data were consistent with an ordered Bi-Bi mechanism for this reaction in which NAD+ is bound first to the enzyme and NADH released last. Kinetic studies indicated that the nucleoside phosphates ATP, ADP and AMP are competitive inhibitors with respect to NAD and noncompetitive inhibitors with respect to S-hydroxymethylglutathione. The inhibitions of the enzyme activity by ATP and ADP are greater at pH 6.0 and 6.5 than at neutral or alkaline pH values. The kinetic studies of formate dehydrogenase (formate:NAD oxidoreductase, EC 1.2.1.2) from the methanol grown C. boidinii suggested also an ordered Bi-Bi mechanism with NAD being the first substrate and NADH the last product. Formate dehydrogenase the last enzyme of the dissimilatory pathway of the methanol metabolism is also inhibited by adenosine phosphates. Since the intracellular concentrations of NADH and ATP are in the range of the Ki values for formaldehyde dehydrogenase and formate dehydrogenase the activities of these main enzymes of the dissimilatory pathway of methanol metabolism in this yeast may be regulated by these compounds.     

Pyruvate dehydrogenase complex of ascites tumour. Activation by AMP and other properties of potential significance in metabolic regulation.

1. AMP is an activator of the pyruvate dehydrogenase complex of the Ehrlich--Lettré ascites tumour, increasing its V up to 2-fold, with Ka of 40 microM at pH 7.4. This activation appears to be an allosteric effect on the decarboxylase subunit of the complex. 2. The pyruvate dehydrogenase complex has a Km for pyruvate within the range 17--36 microM depending on the pH, the optimum pH being approx. 7.4, with a V of approx. 0.1 unit/g of cells. The rate-limiting step is dependent on the transformation of the enzyme--substrate complex. The Km for CoA is 15 microM. The Km for NAD+ is 0.7 mM for both the complex and the lipoamide dehydrogenase. The complex is inhibited by acetyl-CoA competitively with CoA; the Ki is 60 microM. The lipoamide dehydrogenase is inhibited by NADH and NADPH competitively with NAD+, with Ki values of 80 and 90 microM respectively. In the reverse reaction the Km values for NADH and NADPH are essentially equal to their Ki values for the forward reaction, the V for the latter being 0.09 of that of the former. Hence the reaction rate of the complex in vivo is likely to be markedly affected by feedback isosteric inhibition by reduced nicotinamide nucleotides and possibly acetyl-CoA.     

Purification and characterization of a monomeric isocitrate dehydrogenase with dual coenzyme specificity from the photosynthetic bacterium Rhodomicrobium vannielii

An isocitrate dehydrogenase able to function with either NADP or NAD as coenzyme was purified to homogeneity from cell-free extracts of the purple photosynthetic eubacterium Rhodomicrobium vannielii using a rapid two-step procedure involving dye-ligand affinity chromatography. The enzyme was obtained in 60% yield with specific activities of 23 U.mg protein-1 (NADP-linked reaction) and 18.5 U.mg protein-1 (NAD-linked reaction). The purified enzyme was monomeric and migrated with an approximate Mr of 75,000-80,000 on both SDS/PAGE and non-denaturing PAGE. Affinity constants (Km values) of 2.5 microM for NADP and 0.77 mM for NAD and values for kcat/Km of 981,200 min-1.mM-1 (NADP) and 2455 min-1.mM-1 (NAD) indicated a greater specificity for NADP compared to NAD. A number of metabolites were examined for possible differential regulatory effects on the NADP- and NAD-linked reactions, using a dual-wavelength assay. Oxaloacetate was found to be an effective inhibitor of both reactions and the enzyme was also sensitive to concerted inhibition by glyoxylate and oxaloacetate. The amino-acid composition and the identity of 39 residues at the N-terminus were determined and compared to other isocitrate dehydrogenases. The results suggested a relationship between the Rm. vannielii enzyme and the monomeric isocitrate dehydrogenase isoenzyme II from Vibrio ABE-1.     

Formate dehydrogenase. Subunit and mechanism of inhibition by cyanide and azide.

Formate dehydrogenase (EC 1.2.1.2) prepared from peas (Pisum sativum) was a two-subunit enzyme. The enzyme accelerated the formation of an NAD+-cyanide compound having an adsorption band at 330 nm. The enzyme was able to bind one NAD+ molecule per each subunit but only 1 mole of NAD+-cyanide compound was formed per two subunits. The complex of NAD+, cyanide, and the enzyme was very stable and had no catalytic activity. Azide inhibited the formate dehydrogenase reaction in two different ways. By incubation of the enzyme with azide in the presence of NAD+, half of its catalytic activity was lost. The remaining activity was also inhibited by azide but this inhibition was removed competively by formate. Contrary to the case of cyanide the inhibition by azide could be removed by dialysis and no spectral species due to the addition compound of NAD+ and azide could be observed. The data from double recipricol plots of the initial velocity and the formate concentration led to a conclusion that formate dehydrogenase has two sites with about equal catalytic activity. The Km for formate was different for the two catalytic sites (1.67 and 6.25 mM) but the difference was not noticeable in the case of the Km for NAD+.