3-acetylpyridine Adenine Dinucleotide Research Articles

Isotope effect studies of chicken liver NADP malic enzyme: role of the metal ion and viscosity dependence.

The role of the metal ion in the oxidative decarboxylation of malate by chicken liver NADP malic enzyme and details of the reaction mechanism have been investigated by 13C isotope effects. With saturating NADP and the indicated metal ion at a total concentration 10-fold higher than its Km, the following primary 13C kinetic isotope effects at C4 of malate [13(V/Kmal)] were observed at pH 8.0: Mg2+, 1.0336; Mn2+, 1.0365; Cd2+, 1.0366; Zn2+, 1.0337; Co2+, 1.0283; Ni2+, 1.025. Knowing the partitioning of the intermediate oxalacetate between decarboxylation to pyuvate and reduction to malate allows calculation of the intrinsic carbon isotope effect for decarboxylation. For Mg2+ as activator, this was 1.049 with NADP and 1.046 with 3-acetylpyridine adenine dinucleotide phosphate, although the intrinsic primary deuterium isotope effects on dehydrogenation were 5.6 and 4.2, and the partition ratios of the oxalacetate intermediate for decarboxylation as opposed to hydride transfer were 0.11 and 3.96 (the result of the different redox potentials of NADP and the acetylpyridine analogue). The close agreement of the intrinsic 13C isotope effects with each other and with the 13C isotope effect for the Mg2+-catalyzed nonenzymatic decarboxylation of oxalacetate of 1.0489 [Grissom, C. B., & Cleland, W. W. (1986) J. Am. Chem. Soc. 108, 5582] indicates a similarity of transition states for these reactions. It was not possible to calculate reasonable intrinsic carbon isotope effects with the other metal ions by use of the partitioning ratio of oxalacetate because of decarboxylation by another mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

ADP-ribosylation of membrane proteins by bacterial toxins in the presence of NAD glycohydrolase

The ADP-ribosylation of membrane G proteins is difficult to achieve in tissues that are rich in membranebound NAD glycohydrolase (NAD + glycohydrolase, EC 3.2.2.5). For many animal species this problem can be surmounted by inhibiting NAD hydrolysis with a combination of the anti-tuberculous drug, isonicotinic acid hydrazide, and the NAD analog, 3-acetylpyridine adenine dinucleotide, which act synergistically. In their presence, the ADP-ribosylation of cholera and pertussis toxin substrates reach plateau levels even with only 10 μM NAD. Although 3-acetylpyridine adenine dinucleotide acts as a weak substrate for the toxins, it is simple to estimate its contribution to the ADP-ribosylation and thus to determine the total amount of ADP-ribosylation substrate present in a tissue sample. NAD glycohydrolases that are insensitive to isonicotinic acid hydrazide are also less sensitive to 3-acetylpyridine adenine dinucleotide, but may be inactivated by dithiothreitol. Isonicotinic acid hydrazide adenine dinucleotide, the product of an exchange reaction eatalysed by NAD glycohydrolase, runs with NAD in most thin-layer chromatographic systems. It can be separated from NAD, and quantitated if the chromatographic solvent contains benzaldehyde. Isonicotinic acid hydrazide itself inhibits NAD glycohydrolase. It need not first be converted into isonicotinic acid hydrazide adenine dinucleotide.

Energy-linked nicotinamide-nucleotide transhydrogenase. Light-driven transhydrogenase catalyzed by transhydrogenase from beef heart mitochondria reconstituted with bacteriorhodopsin.

Purified nicotinamide-nucleotide transhydrogenase from beef heart mitochondria was co-reconstituted with bacteriorhodopsin to from transhydrogenase-bacteriorhodopsin vesicles that catalyze a 20-fold light-dependent and uncoupler-sensitive stimulation of the reduction of NADP+ and NADP+ analogs by NADH and a 50-fold shift of the nicotinamide nucleotide ratio. In the presence of light, the transhydrogenase-bacteriorhodopsin vesicles catalyzed a pronounced light intensity-dependent inward proton pumping as indicated by a pH shift of the medium. As indicated by pH shifts, proton pumping by the bacteriorhodopsin essentially paralleled the light-driven transhydrogenase. Addition of valinomycin increased the pH shift twice with a concomitant 50% inhibition of the light-driven transhydrogenase, whereas nigericin inhibited the pH shift completely and the light-driven transhydrogenase partially. Taken together, these results suggest that transhydrogenase and bacteriorhodopsin interact through a delocalized proton-motive force. Possible partial reactions of transhydrogenase were investigated with transhydrogenase-bacteriorhodopsin vesicles energized by light. Reduction of oxidized 3-acetylpyridine adenine dinucleotide by NADH, previously claimed to represent partial reactions, was found to require NADPH. Similarly, reduction of thio-NADP+ by NADPH required NADH. It is concluded that these reactions do not represent partial reactions.

Studies of the pH dependence of the formation of binary and ternary complexes with liver alcohol dehydrogenase.

The association of the coenzyme NAD+ to liver alcohol dehydrogenase (LADH) is known to be pH dependent, with the binding being linked to the shift in the pK of some group on the protein from a value of 9-10, in the free enzyme, to 7.5-8 in the LADH-NAD+ binary complex. We have further characterized the nature of this linkage between NAD+ binding and proton dissociation by studying the pH dependence (pH range 6-10) of the proton release, delta n, and enthalpy change, delta Ho(app), for formation of both binary (LADH-NAD+) and ternary (LADH-NAD+-I, where I is pyrazole or trifluoroethanol) complexes. The pH dependence of both delta n and delta Ho(app) is found to be consistent with linkage to a single acid dissociating group, whose pK is perturbed from 9.5 to 8.0 upon NAD+ binding and is further perturbed to approximately 6.0 upon ternary complex formation. The apparent enthalpy change for NAD+ binding is endothermic between pH 7 and pH 10, with a maximum at pH 8.5-9.0. The pH dependence of the delta Ho(app) for both binary and ternary complex formation is consistent with a heat of protonation of -7.5 kcal/mol for the coupled acid dissociating group. The intrinsic enthalpy changes for NAD+ binding and NAD+ plus pyrazole binding to LADH are determined to be approximately 0 and -11.0 kcal/mol, respectively. Enthalpy change data are also presented for the binding of the NAD+ analogues adenosine 5'-diphosphoribose and 3-acetylpyridine adenine dinucleotide.

Kinetics of native and modified liver alcohol dehydrogenase with coenzyme analogues: isomerization of enzyme-nicotinamide adenine dinucleotide complex.

Coenzyme analogues with the adenosine ribose replaced with n-propyl, n-butyl, and n-pentyl groups; coenzyme analogues with the adenosine replaced with 3-(4-acetylanilino)propyl and 6-(4-acetylanilino)hexyl moieties; and nicotinamide mononucleotide, nicotinamide hypoxanthine dinucleotide, and 3-acetylpyridine adenine dinucleotide were used in steady-state kinetic studies with native and activated, amidinated enzymes. The Michaelis and inhibition constants increased up to 100-fold upon modification of coenzyme or enzyme. Turnover numbers with NAD+ and ethanol increased in some cases up to 10-fold due to increased rates of dissociation of enzyme-reduced coenzyme complexes. Rates of dissociation of oxidized coenzyme appeared to be mostly unaffected, but the values calculated (10-60 s-1) were significantly less than the turnover numbers with acetaldehyde and reduced coenzyme (20-900 s-1, at pH 8, 25 degrees C). Rates of association of coenzyme analogues also decreased up to 100-fold. When Lys-228 in the adenosine binding site was picolinimidylated, turnover numbers increased about 10-fold with NAD(H). Furthermore, the pH dependencies for association and dissociation of NAD+ and turnover number with NAD+ and ethanol showed the fastest rates above a pK value of 8.0. Turnover with NADH and acetaldehyde was fastest below a pK value of 8.1. These results can be explained by a mechanism in which isomerization of the enzyme-NAD+ complex (110 s-1) is partially rate limiting in turnover with NAD+ and ethanol (60 s-1) and is controlled by ionization of the hydrogen-bonded system that includes the water ligated to the catalytic zinc and the imidazole group of His-51.

Preparation and characterization of FAD-dependent NADPH-cytochrome P-450 reductase.

NADPH-cytochrome P-450 reductase releases FAD upon dilution into slightly acidic potassium bromide. Chromatography on high performance hydroxylapatite resolved the FAD-dependent reductase from holoreductase. The FAD dependence was matched by a low FAD content, with the ratio of FAD to FMN as low as 0.015. The aporeductase had negligible activity toward cytochrome c, ferricyanide, menadione, dichlorophenolindophenol, nitro blue tetrazolium, and an analogue of NADP, acetylpyridine adenine dinucleotide phosphate. A 4-min incubation in FAD reconstituted from one-half to all of the enzyme activity, as compared to the untreated reductase, depending upon the substrate. After a 2-h reconstitution, the reductase eluted from hydroxylapatite at the same location in the elution profile as did the untreated holoreductase. The reconstituted reductase had little flavin dependence, was nearly equimolar in FMN and FAD, and had close to the specific activity, per mol of flavin, of untreated reductase. The dependence upon FAD implies that FMN is not a competent electron acceptor from NADPH. Thus, the FAD site must be the only point of electron uptake from NADPH.

Characterization of the reduction of 3-acetylpyridine adenine dinucleotide phosphate by benzyl alcohol catalyzed by aldose reductase

Aldose reductase from rat lens catalyzes reduction of the 3-acetylpyridine analog of NADP + by benzyl alcohol; the fluorescence of the reduced pyridine nucleotide produced in this reaction forms the basis of a new, more sensitive assay for this enzyme. The pH dependence and k cat K m ratio of this enzymatic reaction are very similar with NADP + or the analog; however, at pH 7.5, the reaction with the analog has a sevenfold greater k cat. In an NADPH-supported reduction reaction, aldose reductase exhibited similar activity toward benzaldehyde and glyceraldehyde, but benzyl alcohol was a much better substrate than physiological polyols in the analog-dependent oxidation reaction: relative k cat K m ratios were 740 for benzyl alcohol, 2.2 for xylitol, and 1.0 for glycerol. By this assay, a new, high-affinity aldose reductase inhibitor AL-1567 showed noncompetitive inhibition with the pyridine nucleotide analog, with K i = 2.05 × 10 −6 M, and competitive inhibition with benzyl alcohol, with K i = 5.7 × 10 −9 M, indicating that the inhibitor binds the free enzyme with extremely high affinity. Compared to spectrophotometric assays of aldose reductase activity, the fluorometric assay extends the lower limit of detectability of enzyme activity by a factor of 100, and allows the determination of K i values for potent inhibitors of the enzyme with greater accuracy.

30] Snake venom NAD glycohydrolase: Purification, immobilization, and transglycosidation

This chapter presents purification, immobilization, and transglycosidation of snake venom nicotinamide adenine dinucleotide (NAD) glycohydrolase. The enzyme activity can be assayed titrimetrically by measuring the production of hydrogen ion, spectrophotometrically by converting NAD in samples of the reaction mixture to the NAD-cyano adduct absorbing at 327 nm, or spectrophotometricahy by monitoring the 275nm absorbance decrease accompanying the hydrolysis of the alternate substrate, nicotinamide l, N 6 -ethenoadenine dinucleotide (ɛ-NAD). The discontinuous cyanide addition assay is used predominantly for assay of crude preparations of enzyme. One unit of enzyme activity is the amount which catalyzes the hydrolysis of 1 μmol of NAD per minute. All purification steps are performed at 4°. The NADase from Bungarus fasciatus snake venom is adsorbed on Con A-Sepharose and demonstrated to retain both hydrolase and transglycosidase activities in the bound form. The matrix-bound enzyme is stable to repeated washings with buffer and storage at 4° and is used repeatedly for a preparative-scale synthesis of 3-acetylpyridine adenine dinucleotide.

Purification and properties of reconstitutively active nicotinamide nucleotide transhydrogenase of Escherichia coli.

The nicotinamide nucleotide transhydrogenase of Escherichia coli has been purified from cytoplasmic membranes by pre-extraction of the membranes with sodium cholate and Triton X-100, solubilization of the enzyme with sodium deoxycholate in the presence of 1 M potassium chloride, and centrifugation through a 1.1 M sucrose solution. The purified enzyme consists of two subunits, alpha and beta, of apparent Mr 50000 and 47000. During transhydrogenation between NADPH and 3-acetylpyridine adenine dinucleotide by both the purified enzyme reconstituted into liposomes and the membrane-bound enzyme, a pH gradient is established across the membrane as indicated by the quenching of the fluorescence of 9-aminoacridine. Treatment of transhydrogenase with N,N'-dicyclohexylcarbodiimide results in an inhibition of proton pump activity and transhydrogenation, suggesting that proton translocation and catalytic activities are obligatory linked. NADH protected the enzyme against inhibition by N,N'-dicyclohexylcarbodiimide, while NADP, and to a lesser extent NADPH, appeared to increase the rate of inhibition. [14C]Dicyclohexylcarbodiimide preferentially labelled the 50000-Mr subunit of the transhydrogenase enzyme. The presence of an allosteric binding site which reacts with NADH, but not with reduced 3-acetylpyridine adenine dinucleotide, has been demonstrated.

The molecular morphology of bovine heart mitochondrial NADH-ubiquinone reductase. Cross-linking with dithiobis(succinimidyl propionate).

The molecular morphology of NADH-ubiquinone reductase (complex I) was investigated by cross-linking with the cleavable bifunctional reagent, dithiobis(succinimidyl propionate). Cross-linking inhibits the following activities of the complex--NADH----3-acetylpyridine adenine dinucleotide (oxidized), NADH----2,6-dichloroindophenol, NADH----ferricyanide, and NADH----menadione--to different degrees with the greatest inhibition occurring with either ferricyanide or 3-acetylpyridine adenine dinucleotide as electron acceptor. Addition of 150 microM NADH affords partial protection from inhibition. Cross-linking quenches the FMN fluorescence of complex I (288 nm excitation/515 nm emission), and addition of 150 microM NADH greatly reduces the quenching. Treatment of complex I (1 mg/ml) for 2 min with dithiobis(succinimidyl propionate) (0.2 mg/ml) at 4 degrees C revealed a cross-linked product consisting of the following seven subunits: 75-80, 53-57, 42, 33-35, 24-27, 17-18, and 12.5-15.5 kDa. Five minutes of treatment cross-linked the unidentified polypeptides of 69 and 51 kDa to six of the seven complex I subunits, but the 12.5-15.5-kDa subunit may be missing from this cross-linked product, while 15 min of treatment cross-linked additional unidentified polypeptides of 177, 107, 72, and 63 kDa. Since longer times of cross-linking result in a larger number of unidentifiable polypeptide spots, the shorter cross-linking time results are taken as a more accurate picture of the native enzyme conformation. This would indicate that within complex I the following subunits are within 12 A of each other at one or more points in space: 75-80, 53-57, 42-45, 33-35, 24-27, 17-18, and, perhaps, 12.5-15.5 kDa. These subunits represent portions of all three fractions of the enzyme, i.e. flavoprotein, iron-protein, and insoluble or hydrophobic fractions.

Use of intermediate partitioning to calculate intrinsic isotope effects for the reaction catalyzed by malic enzyme.

For those enzymes that proceed via a stepwise reaction mechanism with a discrete chemical intermediate and where deuterium and 13C isotope effects are on separate steps, a new method has been developed to solve for the intrinsic deuterium and 13C kinetic isotope effects that relies on directly observing the partitioning of the intermediate between the forward and reverse directions. This observed partitioning ratio, along with the values of the primary deuterium, tritium, and 13C kinetic isotope effects on V/K for the substrate with the label being followed, allows an exact solution for the intrinsic deuterium and 13C isotope effects, the forward commitment for the deuterium-sensitive step, and the partition ratio for the intermediate in the reaction. This method allows portions of the reaction coordinate diagram to be defined precisely and the relative energy levels of certain activation barriers to be assigned exactly. With chicken liver triphosphopyridine nucleotide (TPN) malic enzyme activated by Mg2+, the partitioning of oxalacetate to pyruvate vs. malate in the presence of TPNH, 0.47, plus previously determined isotope effects gives an intrinsic deuterium isotope effect of 5.7 on hydride transfer and a 13C isotope effect of 1.044 on decarboxylation. Reverse hydride transfer is 10 times faster than decarboxylation, and the forward commitment for hydride transfer is 3.3. The 13C isotope effect is not significantly different with reduced acetylpyridine adenine dinucleotide phosphate replacing TPNH (although the pyruvate/malate partitioning ratio for oxalactate is now 9.9), but replacement of Mg2+ by Mn2+ raises the value to 1.065 (partition ratio 0.99).

Structure of Enoate Reductase from aClostridium tyrobutyricum (C. spec.La1)

Enoate reductase from Clostridium tyrobutyricum was purified by a rapid novel procedure. Chromatography on DEAE-Sepharose and on hydroxyapatite resulted in a high yield of about 90% pure enzyme in less than 10 h. A purity greater than 98% could be obtained by additional chromatography on Sephacryl S-300. The enzyme sediments in the analytical ultracentrifuge as a single, symmetrical boundary with a velocity of S(0)20,w = 24.9 S. Equilibrium ultracentrifugation yielded a molecular mass of 940 000 +/- 20 000 Da. The enzyme contains one type of subunit as shown by dodecyl sulfate electrophoresis and partial sequence determination. A subunit molecular mass of about 73 000 Da was established by dodecyl sulfate electrophoresis and by sedimentation equilibrium analysis in guanidine hydrochloride. In addition to FAD, iron and labile sulfur, the enzyme purified by the new method showed approximately 0.7 mol of FMN per mol of subunit. A dissociation product sedimenting at a velocity of S(0)20,w = 9.8 S can be obtained by various experimental protocols. The fragment was obtained in pure form by gel permeation chromatography. The molecular mass was 230 000 +/- 10 000 Da as shown by sedimentation equilibrium analysis. Thus it appears that the dissociation product is a trimer of the 73 000-Da subunit. The formation of the 10-S fragment by dissociation of the native enzyme is accompanied by the loss of most of the FMN, whereas the FAD content is not changed. The fragment catalysed the reduction of acetylpyridine adenine dinucleotide by NADH. However, enoate reductase activity with NADH or methylviologen as cosubstrate was low. Electron micrographs of negatively stained enoate reductase show trigonal symmetry. The data suggest that enoate reductase is a dodecamer (tetramer of trimers) with tetrahedral symmetry.

Effects of N,N-dicyclohexylcarbodiimide and N-{ethoxycarbonyl}-2-ethoxy-1,2-dihydroquinoline on hydride ion transfer and proton translocation activities of mitochondrial nicotinamide nucleotide transhydrogenase

N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits the mitochondrial energy-linked nicotinamidenucleotide transhydrogenase (TH). Our studies [Phelps, D.C., & Hatefi, Y. (1981) J. Biol. Chem. 256, 8217-8221; Phelps, D.C., & Hatefi, Y. (1984) Biochemistry 23, 4475-4480] suggested that the inhibition site of DCCD is near the NAD(H) binding site, because NAD(H) and competitive inhibitors protected TH against inhibition by DCCD and, unlike the unmodified TH, the DCCD-modified TH did not bind to NAD-agarose. Others [Pennington, R.M., & Fisher, R.R. (1981) J. Biol. Chem. 256, 8963-8969] could not demonstrate protection by NADH, obtained data indicating DCCD inhibits proton translocation by TH much more than hydride ion transfer from NADPH to 3-acetylpyridine adenine dinucleotide (AcPyAD), and concluded that DCCD modifies an essential residue in the proton channel of TH. The present studies show that N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) also inhibits TH. The inhibition is pseudo first order at several EEDQ concentrations, and the reaction order with respect to [EEDQ] is unity, suggesting that inhibition involves the interaction of one molecule of EEDQ with one active unit of TH. The EEDQ-modified TH reacts covalently with [3H]aniline, suggesting that the residue modified by EEDQ is a carboxyl group. More significantly, it has been shown that the absorbance change of oxonol VI at 630 minus 603 nm is a reliable reporter of TH-induced membrane potential formation in submitochondrial particles and that TH-catalyzed hydride ion transfer from NADPH to AcPyAD and the membrane potential induced by this reaction are inhibited in parallel by either DCCD or EEDQ.

The kinetic mechanism of ox liver glutamate dehydrogenase in the presence of the allosteric effector ADP. The oxidative deamination of L-glutamate.

In steady-state kinetic studies of ox liver glutamate dehydrogenase in 0.11 M-potassium phosphate buffer, pH7, at 25 degrees C, the concentration of ADP was varied from 0.5 to 1000 microM. Inhibition was observed except when the concentrations of both glutamate and coenzyme were high, when activation was seen. With NAD+ or NADP+ as coenzyme, 200 microM-ADP was sufficient to saturate the enzyme with respect to the major effect of this nucleotide. In the presence of 210 microM-ADP, widely varied concentrations of coenzyme give linear Lineweaver-Burk plots, in marked contrast with results obtained previously for kinetics without ADP. This has allowed evaluation for the reaction with NAD+, NADP+ and acetylpyridine-adenine dinucleotide (315 microM-ADP in the last case) of all four initial rate parameters, i.e. the phi coefficients in the equation: (Formula: see text) where A is coenzyme and B is glutamate. The relative constancy of phi B and of phi AB/phi A with the different coenzymes point to a compulsory-order mechanism with glutamate as the leading substrate. This conclusion, though unexpected, agrees well with various previous observations on the binding of oxidized coenzyme.

Properties and applications of immobilized snake venom NAD glycohydrolase

The NAD glycohydrolase (NADase) from Bungarus fasciatus snake venom was adsorbed on concanavalin A-Sepharose, and demonstrated to retain both hydrolase and transglycosidase activities in the bound form. The matrix-bound enzyme was stable to repeated washing with buffer and storage at 4°C. The bound enzyme exhibited the same K m value for hydrolysis of nicotinamide-1, N 6-ethenoadenine dinucleotide as previously measured with the soluble, purified form of the enzyme. The bound NADase was used repeatedly for a preparative-scale synthesis of 3-acetylpyridine adenine dinucleotide. It was further demonstrated that the immobilized enzyme could be prepared directly from crude snake venom, thus avoiding the time required for purification. The application of the immobilized snake venom NADase for the preparation of pyridine nucleotide coenzyme analogs has many advantages over procedures used previously for analog synthesis.

Production of lysine by pyruvate dehydrogenase mutants of Brevibacterium flavum.

Mutants with low pyruvate dehydrogenase (PD) activities were derived from a pyruvate kinase-deficient lysine-producing mutant of Brevibacterium flavum, No. 22. They were selected as prototrophic revertants of the acetate auxotrophs of strain No. 22. Among them strain KD-11 produced 55g/liter of lysine as its HCI salt when cultured for 72 hr in a medium containing lOOg/liter of glucose, soybean-meal hydrolysate and methionine. The lysine yield of strain KD-11 was the highest ever reported (55%). The mutant required a higher concentration of methionine for maximum production and gave a smaller amount of cell mass in cultivation than its parent. PD activity of strain No. 22 was stimulated by cysteine, stabilized by glycerol, and gave apparent Kms of 89, 22, 380, 83 μM for pyruvate, coenzyme A, 3-acetylpyridine adenine dinucleotide, and NAD, respectively, under standard conditions. The apparent Km for NAD of PD from strain KD-11 was 10-times higher than that from No. 22. When the concentration of NAD was low,...

Adenosine diphosphoribose transfer reactions catalyzed by Bungarus fasciatus venom NAD glycohydrolase.

Reactions catalyzed by purified Bungarus fasciatus venom NAD glycohydrolase were demonstrated to include ADP-ribose transfer from NAD to alcohols and to imidazole derivatives to produce a variety of ADP-ribosides. The formation of products was monitored by high performance liquid chromatography. In the enzyme-catalyzed alcoholysis of NAD, the ratio of n-alkyl-ADP-riboside formed to the hydrolytic product, ADP-ribose, increased linearly with alcohol concentration. The effectiveness of alcohols as acceptors of the ADP-ribose moiety in these reactions increased with increasing chainlength of the alcohol used. Linear positive chainlength effects extended from methanol to pentanol suggesting facilitation of these reactions by nonpolar interactions. In the methanolysis reaction, NADP, thionicotinamide adenine dinucleotide, nicotinamide-1, N6-ethenoadenine dinucleotide, and 3-acetylpyridine adenine dinucleotide were shown to be as effective as NAD as donor substrates. The NAD glycohydrolase-catalyzed ADP-ribose transfer to pyridine bases to form NAD analogs was studied at pyridine base concentrations above those determined to be saturating for the base exchange reaction. Under these conditions, the ratio of base exchange to hydrolysis of NAD was directly related to the pKa of the ring nitrogen of the pyridine base employed. In addition to alcoholysis and pyridine-base exchange reactions, the snake venom enzyme was demonstrated to catalyze an ADP-ribose transfer reaction to imidazole derivatives. Arginine methyl ester was ineffective as an ADP-ribose acceptor molecule in these reactions.

Energy-linked nicotinamide nucleotide transhydrogenase. Kinetics and regulation of purified and reconstituted transhydrogenase from beef heart mitochondria.

1. Purified and reconstituted nicotinamide nucleotide transhydrogenase from beef heart mitochondria was investigated with respect to kinetic and regulatory properties in uncoupled and coupled liposomes. 2. Double reciprocal plots of initial velocities for the reduction of NAD+ by NADPH versus substrate concentrations were convergent and intersecting on or close to the abscissa, indicating a ternary complex mechanism. The effect of site-specific inhibitors indicates that the order of addition of the substrates to the enzyme is random. 3. Reconstituted transhydrogenase uncoupled by FCCP reveals kinetic properties that are indicative of energization, i.e. an increased and decreased affinity for NADP+ and NAD+, respectively, suggesting that reconstituted transhydrogenase is maintained in an activated conformation. An increased extent of coupling causes a progressively increasing change in the same direction. These results suggest that the uncoupler-dependent enhancement of the rate of reduction of NAD+ by NADPH is due to a decreased Km for NAD+. 4. Reconstituted transhydrogenase catalyzes a transhydrogenation between NADH and 3-acetylpyridine adenine dinucleotide (oxidized) in the presence of NADPH. Reconstituted transhydrogenase also also catalyzes the reduction of thio-NADP+ by NADPH in the presence of NADH. Both reactions are concluded to occur indirectly through the generation of NADP+ and NAD+, respectively, and not directly through a reduced enzyme intermediate. 5. A proton pump mechanism is proposed for transhydrogenase which involves a dimeric form of the enzyme where the two subunits are alternating in proton pumping.

Modification of bovine heart mitochondrial transhydrogenase with tetranitromethane

Modification of pyridine dinucleotide transhydrogenase with tetranitromethane resulted in inhibition of its activity. Development of a membrane potential in submitochondrial particles during the reduction of 3-acetylpyridine adenine dinucleotide (AcPyAD +) by NADPH decreased to nearly the same extent as the transhydrogenase rate on tetranitromethane treatment of the membrane. Kinetics of the inactivation of homogeneous transhydrogenase and the enzyme reconstituted into phosphatidylcholine liposomes indicate that a single essential residue was modified per active monomer. NADP +, NADPH and NADH gave substantial protection against tetranitromethane inactivation of both the nonenergy-linked and energy-linked transhydrogenase reactions of submitochondrial particles and the NADPH → AcPyAD + reaction of reconstituted enzyme. NAD + had no effect on inactivation. Tetranitromethane modification of reconstituted transhydrogenase resulted in a decrease in the rate of coupled H + translocation that was comparable to the decrease in the rate of NADPH → AcPyAD + transhydrogenation. It is concluded that tetranitromethane modification controls the H + translocation process solely through its effect on catalytic activity, rather than through alteration of a separate H +-binding domain. Nitrotyrosine was not found in tetranitromethane-treated transhydrogenase. Both 5,5′-dithiobis(2-nitrobenzoate)-accessible and buried sulfhydryl groups were modified with tetranitromethane. NADH and NADPH prevented sulfhydryl reactivity toward tetranitromethane. These data indicate that the inhibition seen with tetranitromethane results from the modification of a cysteine residue.

Isotopic and kinetic studies and influence of dicoumarol on the soluble hydrogenase from Alcaligenes eutrophus H16

H 2 and NADH oxidation with various acceptors catalyzed by the soluble hydrogenase (hydrogen: NAD + oxidoreductase, EC 1.12.1.2) were investigated in H 2O and 2H 2O. The hydrogenase is active in catalysis between pH 5.5 and 11.5, and the pL optima are remarkably different depending on the acceptor and on the reaction type. In addition to an alkaline shift of about 0.5–0.8 pL units observed for the pL optima of these reactions substituting 2H 2O for H 2O, the H 2 oxidation with NAD, the NADH:acetyipyridine-adenine dinucleotide transhydrogenase reaction and the H 2 evolution with dithionite-reduced methyl viologen revealed kinetic solvent isotope effects of 2.95, 1.25 and 2.0, respectively. For the H 2 oxidation with NAD or methyl viologen (l,l′-dimethyl-4,4′-bipyridinium cation) as the acceptors only a negligible kinetic isotope effect k H 2 / k 2 H 2 of 1.0 to 1.2 was found. This, together with the marked kinetic solvent isotope effect, demonstrates that the H 2-splitting is not the rate-determining step during H 2 uptake with NAD. A k H/ k 3 H of about 2.1–2.5 was observed for the NADH: acetylpyridine-adenine dinucleotide transhydrogenase reaction employing 4S-[4- 3H]NADH. No tritium could be detected in the reduced acetylpyridine-adenine dinucleotide after separating the reaction mixture by chromatography. Hence it is concluded that the transfer of reducing equivalents between the flavin moiety and the pyridine nucleotide is the rate-determining step during hydrogenase catalysis. The hydrogenase catalyzes the tritium transfer from 4S-[4- 3H]NADH into the water also in absence of an electron acceptor. Dicoumarol selectively blocked the H 2 uptake with NAD and rhein as well as the NADH oxidation with acetylpyridine-adenine dinucleotide, ferricyanide and dichlorophenol-indophenol, but did not inhibit the reduction of methyl viologen either with H 2 or with NADH. It is concluded that all the acceptors tested except methyl viologen are bound and reduced at the flavin site. 2-Mercaptoethanol enhanced the rate of H 2 oxidation with methyl viologen as well as protecting the hydrogenase from inactivation with increasing concentrations of the viologen dye. Moreover, methyl viologen was shown to function as an electron acceptor in the H 2-uptake reaction without prior activation of the hydrogenase by catalytic amounts of NAD(P)H. Hydrogenase immobilized on porous glass showed some differences with respect to activation.