Periodate-oxidized 3-aminopyridine adenine dinucleotide phosphate as a fluorescent affinity label for pigeon liver malic enzyme

Treatment of 3-aminopyridine adenine dinucleotide phosphate with sodium periodate resulted in oxidation of the ribose linked to 3-aminopyridine ring and cleavage of the dinucleotide into 3-aminopyridine and adenosine moieties. These two moieties were separated by thin layer chromatography and were synergistically bound to pigeon liver malic enzyme (EC 1.1.1.40), causing inactivation of the enzyme. The inactivation showed saturation kinetics. The apparent binding constant for the reversible enzyme-reagent binary complex (KI) and the maximum inactivation rate constant at saturating reagent concentration (kmax) were found to be 1.1 +/- 0.02 mM and 0.068 +/- 0.001 min-1, respectively. L-Malate at low concentration enhanced the inactivation rate by lowering the KI value whereas high malate concentration increased the kmax. Mn2+ or NADP+ partially protected the enzyme from the inactivation and gave additive protection when used together. L-Malate eliminated the protective effect of NADP+ or Mn2+. Maximum and synergistic protection was afforded by NADP+, Mn2+ plus L-malate (or tartronate). Oxidized and cleaved 3-aminopyridine adenine dinucleotide phosphate was also found to be a competitive inhibitor versus NADP+ in the oxidative decarboxylation reaction catalyzed by malic enzyme with a Ki value of 4.1 +/- 0.1 microM. 3-Aminopyridine adenine dinucleotide phosphate or its periodate-oxidized cleaved products bound to the enzyme anticooperatively. Oxidized 3-aminopyridine adenine dinucleotide phosphate labeled the nucleotide binding site of the enzyme with a fluorescent probe which may be readily traced or quantified. The completely inactivated enzyme incorporated 2 mol of reagent/mol of enzyme tetramer. The inactivation was partially reversible by dilution and could be made irreversible by treating the modified enzyme with sodium borohydride. This fluorescent compound and its counterpart-oxidized 3-aminopyridine adenine dinucleotide may be a potential affinity label for all other NAD(P)+-dependent dehydrogenases.     

Chemical modification of mitochondrial transhydrogenase: evidence for two classes of sulfhydryl groups.

Chemical-modification studies on submitochondrial particle pyridine dinucleotide transhydrogenase (EC 1.6.1.1) demonstrate the presence of one class of sulfhydryl group in the nicotinamide adenine dinucleotide phosphate (NADP) site and another peripheral to the active site. Reaction of the peripheral sulfhydryl group with N-ethylmaleimide, or both classes with 5,5'-dithiobis(2-nitrobenzoic acid), completely inactivated transhydrogenase. NADP+ or NADPH nearly completely protected against 5,5'-dithiobis(2-nitrobenzoic acid) inactivation and modification of both classes of sulfhydryl groups, while NADP+ only partially protected against and NADPH substantially stimulated N-ethylmaleimide inactivation. Methyl methanethiolsulfonate treatment resulted in methanethiolation at both classes of sulfhydryl groups, and either NADP+ or NADPH protected only the NADP site group. S-Methanethio and S-cyano transhydrogenases were active derivatives with pH optima shifted about 1 unit lower than that of the native enzyme. These experiments indicate that neither class of sulfhydryl group is essential for transhydrogenation. Lack of involvement of either sulfhydryl group in energy coupling to transhydrogenation is suggested by the observations that S-methanethio transhydrogenase is functional in (a) energy-linked transhydrogenation promoted by phenazine methosulfate mediated ascorbate oxidation and (b) the generation of a membrane potential during the reduction of NAD+ by reduced nicotinamide adenine dinucleotide phosphate (NADPH).     

Characterization of a pyridine nucleotide-nonspecific glutamate dehydrogenase from Bacteroides thetaiotaomicron.

An oxidized nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide (NADP+/NAD+) nonspecific L-glutamate dehydrogenase from Bacteroides thetaiotaomicron was purified 40-fold (NADP+ or NAD+ activity) over crude cell extract by heat treatment, (NH4)2SO2 fractionation, diethylaminoethyl-cellulose, Bio-Gel A 1.5m, and hydroxylapatite chromatography. Both NADP+- and NAD+-dependent activities coeluted from all chromatographic treatments. Moreover, a constant ratio of NADP+/NAD+ specific activities was demonstrated at each purification step. Both activities also comigrated in 6% nondenaturing polyacrylamide gels. Affinity chromatography of the 40-fold-purified enzyme using Procion RED HE-3B gave a preparation containing both NADP+- and NAD+-linked activities which showed a single protein band of 48,5000 molecular weight after sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The dual pyridine nucleotide nature of the enzyme was most readily apparent in the oxidative direction. Reductively, the enzyme was 30-fold more active with reduced NADP than with reduced NAD. Nonlinear concave 1/V versus 1/S plots were observed for reduced NADP and NH4Cl. Salts (0.1 M) stimulated the NADP+-linked reaction, inhibited the NAD+-linked reaction, and had little effect on the reduced NADP-dependent reaction. The stimulatory effect of salts (NADP+) was nonspecific, regardless of the anion or cation, whereas the degree of NAD+-linked inhibition decreased in the order to I- greater than Br- greater than Cl- greater than F-. Both NADP+ and NAD+ glutamate dehydrogenase activities were also detected in cell extracts from representative strains of other bacteroides deoxyribonucleic acid homology groups.     

Overall kinetic mechanism of saccharopine dehydrogenase (L-glutamate forming) from Saccharomyces cerevisiae

Kinetic studies were carried out for histidine-tagged saccharopine reductase from Saccharomyces cerevisiae at pH 7.0, suggesting a sequential mechanism with ordered addition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to the free enzyme followed by L-alpha-aminoadipate-delta-semialdehyde ( L-AASA) which adds in rapid equilibrium prior to l-glutamate in the forward reaction direction. In the reverse reaction direction, nicotinamide adenine dinucleotide phosphate (NADP) adds to the enzyme followed by addition of saccharopine. Product inhibition by NADP is competitive vs NADPH and noncompetitive vs alpha-AASA and L-glutamate, suggesting that the dinucleotide adds to the free enzyme prior to the aldehyde. Saccharopine is noncompetitive vs NADPH, alpha-AASA, and L-glutamate. In the direction of saccharopine oxidation, NADPH is competitive vs NADP and noncompetitive vs saccharopine, L-glutamate is noncompetitive vs both NADP and saccharopine, while L-AASA is noncompetitive vs saccharopine and uncompetitive vs NADP. The sequential mechanism is also corroborated by dead-end inhibition studies using analogues of AASA, L-glutamate, and saccharopine. 2-Amino-6-heptenoic acid was chosen as a dead-end analogue of L-AASA and is competitive vs AASA, uncompetitive vs NADPH, and noncompetitive vs L-glutamate. alpha-Ketoglutarate (alpha-Kg) serves as the dead-end analogue of L-glutamate and is competitive vs L-glutamate and uncompetitive vs L-AASA and NADPH. In the direction of saccharopine oxidation, N-oxalylglycine, L-pipecolic acid, L-leucine, alpha-ketoglutarate, glyoxylic acid, and L-ornithine were used as dead-end analogues of saccharopine and showed competitive inhibition vs saccharopine and uncompetitive inhibition vs NADP. The equilibrium constant for the reaction was measured at pH 7.0 by monitoring the change in absorbance of NADPH and is 200 M(-1). The value is in good agreement with the value determined using the Haldane relationship.     

Quaternary structure of pigeon liver malic enzyme

Pigeon liver malic enzyme (EC 1.1.1.40) has a double dimer quaternary structure. The NADP+ analogs, aminopyridine adenine dinucleotide phosphate and nicotinamide-1,N6-ethenoadenosine dinucleotide phosphate, bind to the enzyme anti-cooperatively. In the presence of non-cooperative competing ligand NADP+, the binding parameter Hill coefficients of these analogues changed very little. Binding of L-malate with enzyme-AADP+ complex first enhanced then reduced the nucleotide fluorescence. Two L-malate binding sites, with Kd values of 23-30 and 270-400 microM, respectively. for the tight and weak binding sites were postulated. A hybrid model between the sequential and pre-existing asymmetrical models was proposed for the pigeon liver malic enzyme.     

Interactions of nicotinamide-adenine dinucleotide phosphate analogues and fragments with pigeon liver malic enzyme. Synergistic effect between the nicotinamide and adenine moieties.

The structural requirements of the NADP+ molecule as a coenzyme in the oxidative decarboxylation reaction catalysed by pigeon liver malic enzyme were studied by kinetic and fluorimetric analyses with various NADP+ analogues and fragments. The substrate L-malate had little effect on the nucleotide binding. Etheno-NADP+, 3-acetylpyridine-adenine dinucleotide phosphate, and nicotinamide-hypoxanthine dinucleotide phosphate act as alternative coenzymes for the enzyme. Their kinetic parameters were similar to that of NADP+. Thionicotinamide-adenine dinucleotide phosphate, 3-aminopyridine-adenine dinucleotide phosphate, 5'-adenylyl imidodiphosphate, nicotinamide-adenine dinucleotide 3'-phosphate and NAD+ act as inhibitors for the enzyme. The first two were competitive with respect to NADP+ and non-competitive with respect to L-malate; the other inhibitors were non-competitive with NADP+. All NADP+ fragments were inhibitory to the enzyme, with a wide range of affinity, depending on the presence or absence of a 2'-phosphate group. Compounds with this group bind to the enzyme 2-3 orders of magnitude more tightly than those without this group. Only compounds with this group were competitive inhibitors with respect to NADP+. We conclude that the 2'-phosphate group is crucial for the nucleotide binding of this enzyme, whereas the carboxyamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity. There is a strong synergistic effect between the binding of the nicotinamide and adenosine moieties of the nucleotide molecule.     

Kinetic and nuclear magnetic resonance study of the interaction of NADP+ and NADPH with chicken liver fatty acid synthase

The ionic strength dependence of the second-order rate constant for the association of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and chicken liver fatty acid synthase was determined. This rate constant is 7.2 X 10(7) M-1 s-1 at zero ionic strength and 25 degrees C; the effective charge at the cofactor binding sites is +0.8. The conformations of nicotinamide adenine dinucleotide phosphate (NADP+) and NADPH bound to the beta-ketoacyl and enoyl reductase sites were determined from transferred nuclear Overhauser effect measurements. Covalent modification of the enzyme with pyridoxal 5'-phosphate abolished cofactor binding at the enoyl reductase site; this permitted the cofactor conformations at the beta-ketoacyl and enoyl reductase sites to be distinguished. For NADP+ bound to the enzyme, the conformation of the nicotinamide-ribose bond is anti at the enoyl reductase site and syn at the beta-ketoacyl reductase site; the adenine-ribose bond is anti, and the sugar puckers are C3'-endo. Nicotinamide-adenine base stacking was not detected. Structural models of NADP+ at the beta-ketoacyl and enoyl reductase sites were constructed by using the distances calculated from the observed nuclear Overhauser effects. Because of the overlap of the resonances of several nonaromatic NADPH protons with the resonances of HDO and ribose protons, less extensive structural information was obtained for NADPH bound to the enzyme. However, the conformations of NADPH bound to the two reductases are qualitatively the same as those of NADP+, except that the nicotinamide moiety of NADPH is closer to being fully anti at the enoyl reductase site.     

Enzymatic oxidation of NADP+ to its 4-oxo derivative is a side-reaction displayed only by the adrenodoxin reductase type of ferredoxin-NADP+ reductases

We have previously shown that Mycobacterium tuberculosis FprA, an NADPH-ferredoxin reductase homologous to mammalian adrenodoxin reductase, promotes the oxidation of NADP(+) to its 4-oxo derivative 3-carboxamide-4-pyridone adenine dinucleotide phosphate [Bossi RT, Aliverti A, Raimondi D, Fischer F, Zanetti G, Ferrari D, Tahallah N, Maier CS, Heck AJ, Rizzi M et al. (2002) Biochemistry41, 8807-8818]. Here, we provide a detailed study of this unusual enzyme reaction, showing that it occurs at a very slow rate (0.14 h(-1)), requires the participation of the enzyme-bound FAD, and is regiospecific in affecting only the C4 of the NADP nicotinamide ring. By protein engineering, we excluded the involvement in catalysis of residues Glu214 and His57, previously suggested to be implicated on the basis of their localization in the three-dimensional structure of the enzyme. Our results substantiate a catalytic mechanism for 3-carboxamide-4-pyridone adenine dinucleotide phosphate formation in which the initial and rate-determining step is the nucleophilic attack of the nicotinamide moiety by an active site water molecule. Whereas plant-type ferredoxin reductases were unable to oxidize NADP(+), the mammalian adrenodoxin reductase also catalyzed this unusual reaction. Thus, the 3-carboxamide-4-pyridone adenine dinucleotide phosphate formation reaction seems to be a peculiar feature of the mitochondrial type of ferredoxin reductases, possibly reflecting conserved properties of their active sites. Furthermore, we showed that 3-carboxamide-4-pyridone adenine dinucleotide phosphate is good ligand and a competitive inhibitor of various dehydrogenases, making this nucleotide analog a useful tool for the characterization of the cosubstrate-binding site of NADPH-dependent enzymes.     

Comparative aspects of some bacterial dehydrogenases and transhydrogenases

Ragland, T. E. (Brandeis University, Waltham, Mass.), T. Kawasaki, and J. M. Lowenstein. Comparative aspects of some bacterial dehydrogenases and transhydrogenases. J. Bacteriol. 91:236-244. 1966.-Twenty-eight diverse bacterial species were surveyed for the activities and coenzyme specificities of four enzymes: isocitrate dehydrogenase (ICDH), glucose-6-phosphate dehydrogenase (G-6-PDH), 6-phosphogluconate dehydrogenase (6-PGDH), and reduced nicotinamide adenine dinucleotide phosphate-nicotinamide adenine dinucleotide (NAD) transhydrogenase (TH). Most of the species that exhibited a nicotinamide adenine dinucleotide phosphate (NADP)-linked ICDH also showed significant TH activity, but there were several which did not. Only one of the organisms tested, Xanthomonas pruni, had an ICDH active with both NAD and NADP; it was devoid of TH activity. Acetobacter suboxydans, which lacks ICDH altogether, also had no TH. Some of the species examined had G-6-PDH or 6-PGDH (or both) of dual coenzyme specificity, but there was no apparent relation between these findings and the presence or absence of TH. The TH reaction was assayed by use of analogues of NAD as acceptors. The bacteria could be divided into two groups on the basis of TH specificity, one group reacting at a much faster rate with the 3-acetylpyridine analogue of NAD than with the thionicotinamide analogue, whereas the converse was true for the other group. A few organisms showed no marked specificity for either analogue. This division of specificity can be related to the currently accepted taxonomic classification of the organisms, although a few apparent anomalies were found.     

Chemical synthesis of the novel Ca2+ messenger NAADP

The first total chemical synthesis of nicotinamide adenine dinucleotide phosphate (beta-NADP, 2) as a single isomer was achieved. This was subsequently converted into the important second messenger nicotinic acid adenine dinucleotide phosphate (p-NAADP) 1 and the identity of this material confirmed by biological evaluation. This flexible synthetic route offers new opportunities for the generation of NAADP 1 analogues that cannot be generated directly from NADP 2 or mainly enzymatic methods.     

CHEMICAL SYNTHESIS OF THE NOVEL CA2+ MESSENGER NAADP

The first total chemical synthesis of nicotinamide adenine dinucleotide phosphate (β-NADP, 2) as a single isomer was achieved. This was subsequently converted into the important second messenger nicotinic acid adenine dinucleotide phosphate (β-NAADP) 1 and the identity of this material confirmed by biological evaluation. This flexible synthetic route offers new opportunities for the generation of NAADP 1 analogues that cannot be generated directly from NADP 2 or mainly enzymatic methods.     

Photosynthetic response of transgenic soybean plants, containing an Arabidopsis P5CR gene, during heat and drought stress

The biochemical basis of heat/drought tolerance was investigated by comparing the response of antisense and sense transgenic soybean plants (containing the L-delta1-pyrroline-5-carboxylate reductase gene) with non-transgenic wild-type plants. The plants were subjected to a simultaneous drought and heat stress of 2 days, whereafter they were rewatered at 25 degrees C. During this time the sense plants only showed mild symptoms of stress compared to the antisense plants which were severely stressed. Upon stress, nicotinamide adenine dinucleotide phosphate (NADP+) levels decreased in antisense while it increased in sense plants. Recovery with respect to NADP+ levels was best in sense plants. Sense plants had the highest ability to accumulate proline during stress and to metabolise proline after rewatering. Analyses of the fast phase chlorophyll-a fluorescence transients showed dissociation of the oxygen-evolving complex (OEC) upon stress in all plants tested. In the sense plants, which best resisted the stress, OEC dissociation was bypassed by proline feeding electrons into photosystem 2 (PSII), maintaining an acceptable nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) level, preventing further damage. Upon recovery, NADPH is consumed during oxidation of accumulated proline providing high Levels of NADP+ to act as electron acceptor to PSII, which indirectly may ameliorate the inhibition and/or the effect of uncoupling of the OEC.     

Relationship between pyridine nucleotide levels and ribonucleotide reductase activity in yoshida ascites hepatoma AH 130

We measured both pyridine nucleotide levels and ribonucleotide reductase-specific activity in Yoshida ascites hepatoma cells as a function of growth in vivo and during recruitment from non-cycling to cycling state in vitro. Oxidized nicotinamide adenine dinucleotide (NAD⁺) and reduced nicotinamide adenine dinucleotide (NADP) levels remained unchanged during tumour growth, while NADP⁺ and reduced nicotinamide adenine dinucleotide phosphate (NADPH) levels were very high in exponentially growing cells and markedly decreased in the resting phase. Ribonucleotide reductase activity paralleled NADP(H) (NADP⁺ plus NADPH) intracellular content. The concomitant increase in both NADP(H) levels and ribonucleotide reductase activity was also observed during G1-S transition in vitro. Cells treated with hydroxyurea showed a comparable correlation between the pool size of NADP(H) and ribonucleotide reductase activity. On the basis of these findings, we suggest that fluctuations in NADP(H) levels and ribonucleotide reductase activity might play a critical role in cell cycle regulation.     

Regulation of the nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenases of Saccharomyces cerevisiae

Saccharomyces cerevisiae contains two distinct l-glutamate dehydrogenases. These enzymes are affected in a reciprocal fashion by growth on ammonia or dicarboxylic amino acids as the nitrogen source. The specific activity of the nicotinamide adenine dinucleotide phosphate (NADP) (anabolic) enzyme is highest in ammonia-grown cells and is reduced in cells grown on glutamate or aspartate. Conversely, the specific activity of the nicotinamide adenine dinucleotide (NAD) (catabolic) glutamate dehydrogenase is highest in cells grown on glutamate or aspartate and is much lower in cells grown on ammonia. The specific activity of both enzymes is very low in nitrogen-starved yeast. Addition of the ammonia analogue methylamine to the growth medium reduces the specific activity of the NAD-dependent enzyme and increases the specific activity of the NADP-dependent enzyme.     

Properties of Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum

Li, Lan-Fun (Western Reserve University School of Medicine, Cleveland, Ohio), Lars Ljungdahl, and Harland G. Wood. Properties of nicotinamide adenine dinucleotide phosphate-dependent formate dehydrogenase from Clostridium thermoaceticum. J. Bacteriol. 92: 405-412. 1966.-A nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase has been isolated from C. thermoaceticum. The enzyme is very sensitive to oxygen and requires sulfhydryl compounds for activity. The apparent K(m) at 50 C and pH 7.0 for NADP is 5.9 x 10(-5)m and for formate, 2.2 x 10(-4)m. The enzyme is most active at about 60 C and at pH values between 7.0 and 9.0. The enzyme catalyzes an exchange between C(14)O(2) and formate, which requires NADP, but net synthesis of formate from CO(2) and reduced nicotinamide adenine dinucleotide phosphate could not be demonstrated. The reaction does not involve ferredoxin.     

Conformational change of Arabidopsis thaliana thioredoxin reductase after binding of pyridine nucleotide and thioredoxin

We have found that the binding of NADP+ (Kd = 0.86+/-0.11 microM) enhanced the FAD fluorescence of Arabidopsis thaliana NADPH:thioredoxin reductase (TR, EC 1.6.4.5) by 2 times, whereas the binding of 3-aminopyridine adenine dinucleotide phosphate (AADP+) (Kd < 0.1 microM) quenched the fluorescence by 20%. Thioredoxin (TRX) also enhanced the FAD fluorescence by 35%. The Kd of TR-NADP+ and TR-AADP+ complexes did not change in the presence of 45 microM TRX. Our findings imply that the binding of NADP+ and AADP+ at the NADP(H)-binding site of A. thaliana TR, and/or the binding of TRX in the vicinity of the catalytic disulfide increase the content of fluorescent FR conformer (NADP(H)-binding site adjacent to flavin). The different effects of NADP+ and AADP+ on FAD fluorescence intensity may be explained by the superposition of two opposite factors: i) increased content of fluorescent FR conformer upon binding of NADP+ or AADP+; ii) quenching of FAD fluorescence by electron-donating 3-aminopyridinium ring of AADP+.     

Single-sample preparation for simultaneous cellular redox and energy state determination

A simple and reliable method for the preparation of biological samples for the evaluation of biochemical parameters representative of the redox and energy states, such as glutathione (GSH), oxidized glutathione (GSSG), oxidized nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide phosphate (NADPH), coenzyme A (CoASH), oxidized CoASH, ascorbate, malondialdehyde, oxypurines, nucleosides, and energy metabolites, is presented. Fast deproteinization under nonoxidizing conditions is obtained by tissue homogenization in ice-cold, nitrogen-saturated CH3CN + 10 mM KH2PO4 (3:1; v:v), pH 7.40. After sample centrifugation to pellet precipitated proteins, organic solvent removal is performed on clear supernatants by three washings with large volumes of high-performance liquid chromatography (HPLC)-grade chloroform. The remaining aqueous phase, free of solvent and any lipid-soluble substances that may interfere with the further metabolite analysis, is used for the simultaneous ion-pairing HPLC determination of 39 compounds by means of a Kromasil C-18, 250 x 4.6-mm, 5-microm-particle-size column with tetrabutylammonium hydroxide as the pairing reagent. Results obtained by using the present method to prepare different rat tissue extracts demonstrate that it is possible to perform a single tissue preparation only for monitoring, in the same sample, compounds representative of the redox state (through the direct determination of GSH, GSSG, NAD+, NADH, NADP+, NADPH, CoASH, and oxidized CoASH) and of the cell energy state (by the analysis of oxypurines, nucleosides, and energy metabolites). Applicability of this sample processing procedure to quantify variations of the aforementioned compounds under pathological conditions was effected in rats subjected to moderate closed-head trauma.     

Factor 420-dependent tyridine nucleotide-linked hydrogenase system of Methanobacterium ruminantium.

Methanobacterium ruminantium was shown to possess a nicotinamide adenine dinucleotide phosphate (NADP)-linked factor 420 (F420)-dependent hydrogenase system. This system was also shown to be present in Methanobacterium strain MOH. The hydrogenase system of M. ruminantium also links directly to F420, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), methyl viologen, and Fe-3 plus. It has a pH optimum of about 8 and an apparent Km for F420 of about 5 x 10-6 M at pH 8 when NADP is the electron acceptor. The F420-NADP oxidoreductase activity is inactive toward nicotinamide adenine dinucleotide (nad) and no NADPH:NAD or FADH2(FMNH2):NAD transhydrogenase system was detected. Neither crude ferredoxin nor boiled crude extract of Clostridium pasteuranum could replace F420 in the NADP-linked hydrogenase reaction of M. ruminantium. Also, neitther F420 nor a curde "ferredoxin" fraction from M. ruminantium extracts could substitute for ferredoxin in the pyruvate-ferredoxin oxidoreductase reaction of C. pasteurianum.     

NADPH-cytochrome P-450 oxidoreductase. The role of cysteine 566 in catalysis and cofactor binding

Site-directed mutagenesis was employed to investigate the role of Cys566 in the catalytic mechanism of rat liver NADPH-cytochrome P-450 oxidoreductase. Rat NADPH-cytochrome P-450 oxidoreductase and mutants containing either alanine or serine at position 566 were expressed in Escherichia coli and purified to homogeneity. Substitution of alanine at position 566 had no effect on enzymatic activity with the acceptors cytochrome c and ferricyanide but did increase trans-hydrogenase activity with 3-acetylpyridine adenine dinucleotide phosphate by 79%. The Km for NADPH was increased 2.5-fold, and the NADP+ KI was increased 4.8-fold compared with that found for the wild-type enzyme. The conservative substitution, Ser566, produced a 50% decrease in cytochrome c reductase activity whereas activity with ferricyanide was decreased 57%, and 3-acetylpyridine adenine dinucleotide phosphate activity was unaffected. The NADPH Km was increased 4.6-fold, and the NADP+ KI increased 7.6-fold. The dependence of cytochrome c reductase activity on the KCl concentration was markedly altered by the Cys566 substitutions. Maximum activity for the wild-type enzyme was observed at approximately 0.18 M KCl whereas maximum activity for the mutant enzymes was observed between 0.04 and 0.09 M KCl. The pH dependence of cytochrome c reductase activity, cytochrome c Km, and flavin content were unaffected by these substitutions. These results demonstrate that Cys566 is not essential for activity of rat liver NADPH-cytochrome P-450 oxidoreductase although the cysteine side chain does affect the interaction of NADPH with the enzyme.