Malate dehydrogenase of the cytosol. A kinetic investigation of the reaction mechanism and a comparison with lactate dehydrogenase.

1. The mechanisms of the reduction of oxaloacetate and of 3-fluoro-oxaloacetate by NADH catalysed by cytoplasmic pig heart malate dehydrogenase (MDH) were investigated. 2. One mol of dimeric enzyme produces 1.7+/-0.4 mol of enzyme-bound NADH when mixed with saturating NAD+ and L-malate at a rate much higher than the subsequent turnover at pH 7.5. 3. Transient measurements of protein and nucleotide fluorescence show that the steady-state complex in the forward direction is MDH-NADH and in the reverse direction MDH-NADH-oxaloacetate. 4. The rate of dissociation of MDH-NADH was measured and is the same as Vmax. in the forward direction at pH 7.5. Both NADH-binding sites are kinetically equivalent. The rate of dissociation varies with pH, as does the equilibrium binding constant for NADH. 5. 3-Fluoro-oxaloacetate is composed of three forms (F1, F2 and S) of which F1 and F2 are immediately substrates for the enzyme. The third form, S, is not a substrate, but when the F forms are used up form S slowly and non-enzymically equilibrates to yield the active substrate forms. S is 2,2-dihydroxy-3-fluorosuccinate. 6. The steady-state compound during the reduction of form F1 is an enzyme form that does not contain NADH, probably MDH-NAD+-fluoromalate. The steady-state compound for form F2 is an enzyme form containing NADH, probably MDH-NADH-fluoro-oxaloacetate. 7. The rate-limiting reaction in the reduction of form F2 shows a deuterium isotope rate ratio of 4 when NADH is replaced by its deuterium analogue, and the rate-limiting reaction is concluded to be hydride transfer. 8. A novel titration was used to show that dimeric cytoplasmic malate dehydrogenase contains two sites that can rapidly reduce the F1 form of 3-fluoro-oxaloacetate. The enzyme shows 'all-of-the-sites' behaviour. 9. Partial mechanisms are proposed to explain the enzyme-catalysed transformations of the natural and the fluoro substrates. These mechanisms are similar to the mechanism of pig heart lactate dehydrogenase and this, and the structural results of others, can be explained if the two enzymes are a product of divergent evolution.     

Pig liver pyruvate carboxylase. Purification, properties and cation specificity

1. Pyruvate carboxylase was purified to apparent homogeneity from pig liver mitochondria and shown to be free of all kinetically contaminating enzymes. 2. The enzyme has a mol. wt. of 520000 and is composed of four subunits, each with a mol. wt. of 130000. 3. The enzyme can exist as the active tetramer, dimer and monomer, although the tetramer appears to be the form in which the enzyme is normally assayed. 4. For every 520000g of the enzyme there are 4mol of biotin, 3mol of zinc and 1mol of magnesium. No significant concentrations of manganese were detected. 5. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicates three polypeptide chains per monomer unit, each with a mol. wt. of 47000. 6. The amino acid analysis, stoicheiometry of the reaction and the activity of the enzyme as a function of pH are also presented. 7. The enzyme is activated by a variety of univalent cations but not by Tris(+) or triethanolamine(+). 8. The activity of the enzyme is dependent on the presence of acetyl-CoA; the low rate in the absence of added acetyl-CoA is not due to an enzyme-bound acyl-CoA. The dissociation constant for enzyme-bound acetyl-CoA is a marked function of pH.     

Coenzyme binding by 3-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme: lecithin acts as an allosteric modulator to enhance the affinity for coenzyme

The role of phospholipid in the binding of coenzyme, NAD(H), to 3-hydroxybutyrate dehydrogenase, a lipid-requiring membrane enzyme, has been studied with the ultrafiltration binding method, which we optimized to quantitate weak ligand binding (KD in the range 10-100 microM). 3-Hydroxybutyrate dehydrogenase has a specific requirement of phosphatidylcholine (PC) for optimal function and is a tetramer quantitated both for the apodehydrogenase, which is devoid of phospholipid, and for the enzyme reconstituted into phospholipid vesicles in either the presence or absence of PC. We find that (i) the stoichiometry for NADH and NAD binding is 0.5 mol/mol of enzyme monomer (2 mol/mol of tetramer); (ii) the dissociation constant for NADH binding is essentially the same for the enzyme reconstituted into the mixture of mitochondrial phospholipids (MPL) (KD = 15 +/- 3 microM) or into dioleoyl-PC (KD = 12 +/- 3 microM); (iii) the binding of NAD+ to the enzyme-MPL complex is more than an order of magnitude weaker than NADH binding (KD approximately 200 microM versus 15 microM) but can be enhanced by formation of a ternary complex with either 2-methylmalonate (apparent KD = 1.1 +/- 0.2 microM) or sulfite to form the NAD-SO3- adduct (KD = 0.5 +/- 0.1 microM); (iv) the binding stoichiometry for NADH is the same (0.5 mol/mol) for binary (NADH alone) and ternary complexes (NADH plus monomethyl malonate); (v) binding of NAD+ and NADH together totals 0.5 mol of NAD(H)/mol of enzyme monomer, i.e., two nucleotide binding sites per enzyme tetramer; and (vi) the binding of nucleotide to the enzyme reconstituted with phospholipid devoid of PC is weak, being detected only for the NAD+ plus 2-methylmalonate ternary complex (apparent KD approximately 50 microM or approximately 50-fold weaker binding than that for the same complex in the presence of PC). The binding of NADH by equilibrium dialysis or of spin-labeled analogues of NAD+ by EPR spectroscopy gave complementary results, indicating that the ultrafiltration studies approximated equilibrium conditions. In addition to specific binding of NAD(H) to 3-hydroxybutyrate dehydrogenase, we find significant binding of NAD(H) to phospholipid vesicles. An important new finding is that the nucleotide binding site is present in 3-hydroxybutyrate dehydrogenase in the absence of activating phospholipid since (a) NAD+, as the ternary complex with 2-methylmalonate, binds to the enzyme reconstituted with phospholipid devoid of PC and (b) the apodehydrogenase, devoid of phospholipid, binds NADH or NAD-SO3- weakly (half-maximal binding at approximately 75 microM NAD-SO3- and somewhat weaker binding for NADH).(ABSTRACT TRUNCATED AT 400 WORDS)     

Half-site reactivity of an essential thiol group of D-beta-hydroxybutyrate dehydrogenase

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme, which is a tetramer both in the mitochondrial inner membrane and as the purified enzyme reconstituted with phospholipid. For the active enzyme-phospholipid complex in the absence of ligands, we previously found that reaction with N-ethylmaleimide (at 5 mol/mol of enzyme subunit) resulted in progressive loss of enzymic activity with an inactivation stoichiometry of 1 equiv of sulfhydryl derivatized per mole of enzyme and a maximum derivatization of 2 equiv [Latruffe, N., Brenner, S. C., & Fleischer, S. (1980) Biochemistry 19, 5285-5290]. We now find, in the presence of nucleotide or substrate, that the rate of inactivation is significantly reduced, which indicates that these ligands afford protection of the essential sulfhydryl. Further, in the presence of ligands, the inactivation stoichiometry is 0.5, consistent with half-of-the-site reactivity of the essential sulfhydryl. Thus, at a low ratio of N-ethylmaleimide to enzyme, nucleotide or substrate affords essentially complete protection of the nonessential sulfhydryl from derivatization. The binding characteristics of NADH to both the native and N-ethylmaleimide-derivatized enzyme have been compared by fluorescence spectroscopy. Quenching of intrinsic tryptophan fluorescence of the protein shows that the enzyme, derivatized with N-ethylmaleimide either in the absence or in the presence of NAD+, binds NADH but with a reduced Kd (approximately 50 microM as compared with approximately 20 microM for native enzyme). However, a critical change has occurred in that resonance energy transfer from protein to bound NADH, observed in the native enzyme, is abolished in the N-ethylmaleimide-derivatized enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

The identification of intermediates in the reaction of pig heart lactate dehydrogenase with its substrates

Pig heart lactate dehydrogenase was studied in the direction of pyruvate and NADH formation by recording rapid changes in extinction, proton concentration, nucleotide fluorescence and protein fluorescence. Experiments measuring extinction changes show that there is a very rapid formation of NADH within the first millisecond and that the amplitude of this phase (phase 1) increases threefold over the pH range 6-8. A second transient rate (phase 2) can also be distinguished (whose rate is pH-dependent), followed by a steady-state rate (phase 3) of NADH production. The sum of the amplitudes of the first two phases corresponds to 1mol of NADH produced/mol of active sites of lactate dehydrogenase. Experiments that measured the liberation of protons by using Phenol Red as an indicator show that no proton release occurs during the initial very rapid formation of NADH (phase 1), but protons are released during subsequent phases of NADH production. Fluorescence experiments help to characterize these phases, and show that the very rapid phase 1 corresponds to the establishment of an equilibrium between E(NAD) (Lactate) right harpoon over left harpoon H(+)E(NADH) (Pyruvate). This equilibrium can be altered by changing lactate concentration or pH, and the H(+)E(NADH) (Pyruvate) species formed has very low nucleotide fluorescence and quenched protein fluorescence. Phase 2 corresponds to the dissociation of pyruvate and a proton from the complex with a rate constant of 1150s(-1). The observed rate constant is slower than this and is proportional to the position of the preceding equilibrium. The E(NADH) formed has high nucleotide fluorescence and quenched protein fluorescence. The reaction, which is rate-limiting during steady-state turnover, must then follow this step and be involved with dissociation of NADH from the enzyme or some conformational change immediately preceding dissociation. Several inhibitory complexes have also been studied including E(NAD+) (Oxamate) and E(NADH) (Oxamate') and the abortive ternary complex E(NADH) (Lactate). The rate of NADH dissociation from the enzyme was measured and found to be the same whether measured by ligand displacement or by relaxation experiments. These results are discussed in relation to the overall mechanism of lactate dehydrogenase turnover and the independence of the four binding sites in the active tetramer.     

NADH oxidase from the extreme thermophile Thermus aquaticus YT-1. Purification and characterisation

A protein with NADH oxidase activity from the extreme thermophile Thermus aquaticus YT-1 was purified and characterised. The enzyme was found to have a relative molecular mass of 110,000 and be composed of two subunits of identical size. FAD was found to be present at a concentration of 0.7 mol/mol dimer and was required for activity. During the oxidation of NADH, oxygen uptake takes place with the production of hydrogen peroxide. The enzyme had, with the exception of a higher glutamic acid and tryptophan content, a similar amino acid composition as the NADH oxidase isolated from the mesophile Bacillus megaterium. Purified NADH oxidase was found to have a Km of 39 microM for beta-NADH and a Vmax of 4.68 mumol NADH mg-1 min-1 and was still active at 95 degrees C. Enzymatic activity was found to be independent of pH between 5.0 and 10.5.     

Activation of L-lysine epsilon-dehydrogenase from Agrobacterium tumefaciens by several amino acids and monocarboxylates

The activation of lysine epsilon-dehydrogenase [EC 1.4.1.] by L-lysine was dependent on lysine concentration and was accompanied by association of the dimeric enzymes to a tetramer. The lysine concentration required for the half-maximal activation was 0.28 mM, which was lower than the Km value for L-lysine. In addition to L-lysine, several compounds, which were neither substrates nor inhibitors, activated the enzyme. The compounds which activated the enzyme have common structural characteristics: they have both a carboxyl group and a hydrophobic side chain. These activators also induced the association of the enzyme. The activation of the enzyme occurred well over the pH range 5.0 to 7.5, and the maximal activation was obtained by preincubation for 5 min at 30 degrees C and pH 7.4, when 5 mM L-lysine or 6-aminocaproate was used as an activator. NADH binding experiments indicated that about 2 mol of NADH bind to 1 mol of the tetrameric enzyme: the dimeric enzyme has one catalytic site. Binding experiments with n-[1-14C]heptanoate and L-[U-14C]lysine showed that approximately 2 mol of ligands bind to 1 mol of the dimeric enzyme and L-lysine could not bind to the catalytic site of the enzyme in the absence of NAD+. These results indicate the presence of one catalytic site and two activator binding binding sites in the dimeric enzyme.     

Malate dehydrogenase of the cytosol. Preparation and reduced nicotinamide–adenine dinucleotide-binding studies

1. Two methods of preparing pig heart soluble malate dehydrogenase are described. A slow method yields an enzyme composed of three electrophoretically separable subforms. The more rapid method reproducibly gives a high yield of an enzyme that consists predominantly of the least acid subform. 2. The A(1%) (1cm) of the protein was redetermined as 15 at 280nm. By using this value the enzyme molecule was found to contain two independent and indistinguishable NADH-binding sites in titrations with NADH. 3. No evidence was found for the dissociation of the enzyme in the concentration range 0.02-7.2mum. 4. l-Malate (0.1m) tightened the binding of NADH to both pig and ox heart enzyme (2-fold), but, in contrast with the report by Mueggler, Dahlquist & Wolfe [(1975) Biochemistry14, 3490-3497], did not cause co-operative interactions between the binding sites. 5. Fructose 1,6-bisphosphate had no effect on the binding of NADH to the pig heart enzyme, but with the ox heart enzyme the NADH is slowly oxidized. This slow oxidation explains the ;sigmoidal' binding curves obtained when NADH was added to ox heart soluble malate dehydrogenase in the presence of fructose 1,6-bisphosphate [Cassman (1973) Biochem. Biophys. Res. Commun.53, 666-672] without the postulate of site-site interactions. 6. It is concluded that neither l-malate nor fructose 1,6-bisphosphate could in vivo modulate the activity of soluble malate dehydrogenase and alter the rates of transport of NADH between the cytosol and the mitochondrion. 7. Details of the preparation of soluble malate dehydrogenase have been deposited as Supplementary Publication SUP 50080 (8 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1978) 169, 5.     

Purification and characterisation of the NADH:acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWW0 of Pseudomonas putida mt-2.

The xylene monooxygenase system encoded by the TOL plasmid pWW0 of Pseudomonas putida catalyses the hydroxylation of a methyl side-chain of toluene and xylenes. Genetic studies have suggested that this monooxygenase consists of two different proteins, products of the xylA and xylM genes, which function as an electron-transfer protein and a terminal hydroxylase, respectively. In this study, the electron-transfer component of xylene monooxygenase, the product of xylA, was purified to homogeneity. Fractions containing the xylA gene product were identified by its NADH:cytochrome c reductase activity. The molecular mass of the enzyme was determined to be 40 kDa by SDS/PAGE, and 42 kDa by gel filtration. The enzyme was found to contain 1 mol/mol of tightly but not covalently bound FAD, as well as 2 mol/mol of non-haem iron and 2 mol/mol of acid-labile sulfide, suggesting the presence of two redox centers, one FAD and one [2Fe-2S] cluster/protein molecule. The oxidised form of the protein had absorbance maxima at 457 nm and 390 nm, with shoulders at 350 nm and 550 nm. These absorbance maxima disappeared upon reduction of the protein by NADH or dithionite. The NADH:acceptor reductase was capable of reducing either one- or two-electron acceptors, such as horse heart cytochrome c or 2,6-dichloroindophenol, at an optimal pH of 8.5. The reductase was found to have a Km value for NADH of 22 microM. The oxidation of NADH was determined to be stereospecific; the enzyme is pro-R (class A enzyme). The titration of the reductase with NADH or dithionite yielded three distinct reduced forms of the enzyme: the reduction of the [2Fe-2S] center occurred with a midpoint redox potential of -171 mV; and the reduction of FAD to FAD. (semiquinone form), with a calculated midpoint redox potential of -244 mV. The reduction of FAD. to FAD.. (dihydroquinone form), the last stage of the titration, occurred with a midpoint redox potential of -297 mV. The [2Fe-2S] center could be removed from the protein by treatment with an excess of mersalyl acid. The [2Fe-2S]-depleted protein was still reduced by NADH, giving rise to the formation of the anionic flavin semiquinone observed in the native enzyme, thus suggesting that the electron flow was NADH --> FAD --> [2Fe-2S] in this reductase. The resulting protein could no longer reduce cytochrome c, but could reduce 2,6-dichloroindophenol at a reduced rate.     

9-(5',6'-dideoxy-beta-D-ribo-hex-5'-ynofuranosyl)adenine, a novel irreversible inhibitor of S-adenosylhomocysteine hydrolase

The acetylenic analogue of adenosine 9-(5',6'-dideoxy-beta-D-ribo-hex-5'-ynofuranosyl)adenine has been synthesized, and its behavior as an inhibitor of bovine S-adenosylhomocysteine hydrolase has been examined. Incubation of the enzyme with excess inhibitor caused a time-dependent, irreversible inactivation of the enzyme that was accompanied by the reduction of two equivalents of NAD+ to NADH and the loss of the two remaining equivalents of NAD+. With use of radiolabeled inhibitor, it was established that 4 equiv of the acetylenic analog bind irreversibly to the enzyme and that 4 equiv were required to inactivate the enzyme completely. The inactivated enzyme could not be reactivated by incubation with NAD+. Denaturation studies revealed that 2 equiv of the inhibitor are bound more tightly to the enzyme than the remainder, suggesting the formation of a covalent linkage between the oxidized inhibitor and the enzyme. The putative covalent linkage was found to be acid sensitive but stable to mild base. The linkage could not be stabilized by treatment of the enzyme-inhibitor complex with either borohydride or cyanoborohydride. A Kl of 173 nM was measured for the inhibitor, making it one of the more potent inhibitors that have been reported. The enzyme used in these studies was isolated by modification of an affinity chromatography method reported by Narayanan and Borchardt [(1988) Biochim. Biophys. Acta 965, 22-28]. The affinity chromatography unexpectedly led to the isolation of two forms of the enzyme. The major form contained 4.0 mol of nucleotide cofactor/mol of enzyme tetramer, while the minor form carried only 2.0 mol/tetramer.     

Methanoarchaeal sulfolactate dehydrogenase: prototype of a new family of NADH-dependent enzymes

The crystal structure of the sulfolactate dehydrogenase from the hyperthermophilic and methanogenic archaeon Methanocaldococcus jannaschii was solved at 2.5 A resolution (PDB id. 1RFM). The asymmetric unit contains a tetramer of tight dimers. This structure, complexed with NADH, does not contain a cofactor-binding domain with 'Rossmann-fold' topology. Instead, the tertiary and quaternary structures indicate a novel fold. The NADH is bound in an extended conformation in each active site, in a manner that explains the pro-S specificity. Cofactor binding involves residues belonging to both subunits within the tight dimers, which are therefore the smallest enzymatically active units. The protein was found to be a homodimer in solution by size-exclusion chromatography, analytical ultracentrifugation and small-angle neutron scattering. Various compounds were tested as putative substrates. The results indicate the existence of a substrate discrimination mechanism, which involves electrostatic interactions. Based on sequence homology and phylogenetic analyses, several other enzymes were classified as belonging to this novel family of homologous (S)-2-hydroxyacid dehydrogenases.     

NADH oxidation by the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae: functional role of the NqrF subunit

The Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae is a six subunit enzyme containing four flavins and a single motif for the binding of a Fe-S cluster on its NqrF subunit. This study reports the production of a soluble variant of NqrF (NqrF') and its individual flavin and Fe-S-carrying domains using V. cholerae or Escherichia coli as expression hosts. NqrF' and the flavin domain each contain 1 mol of FAD/mol of enzyme and exhibit high NADH oxidation activity (20,000 micromol min(-1) mg(-1)). EPR, visible absorption, and circular dichroism spectroscopy indicate that the Fe-S cluster in NqrF' and its Fe-S domain is related to 2Fe ferredoxins of the vertebrate-type. The addition of NADH to NqrF' results in the formation of a neutral flavosemiquinone and a partial reduction of the Fe-S cluster. The NqrF subunit harbors the active site of NADH oxidation and acts as a converter between the hydride donor NADH and subsequent one-electron reaction steps in the Na(+)-translocating NADH:quinone oxidoreductase complex. The observed electron transfer NADH --> FAD --> [2Fe-2S] in NqrF requires positioning of the FAD and the Fe-S cluster in close proximity in accordance with a structural model of the subunit.     

The use of ternary complexes to study ionizations and isomerizations during catalysis by lactate dehydrogenase

1. The binding of oxamate to pig heart and pig muscle isoenzymes of lactate dehydrogenase in the presence of NADH was studied by fluorescence titration. The dissociation constant of oxamate from the heart enzyme complex is 3mum and from the muscle isoenzyme 25mum at pH5. These values quantitatively increase with pH as predicted if oxamate can bind only to the enzyme-NADH complex if a group with pK6.9 is protonated. There are four non-interacting oxamate-binding sites per tetramer. 2. o-Nitrophenylpyruvate is a poor substrate for both isoenzymes but has a reasonable affinity to the heart isoenzyme. Initially, it forms an enzyme-NADH-substrate complex, which can be detected either by protein-fluorescence quenching or by NADH-fluorescence quenching. The pH-dependence of the dissociation constant of nitrophenylpyruvate also shows that this ternary complex can only form if a group with pK6.8 is protonated. Taken with the results of chemical-modification experiments, these results allow the pK of 6.8 to be assigned to a system probably involving the imidazole side chain of histidine-195. Formation of a ternary complex from a binary one at pH8 is predicted to result in a proton being taken up from solution. 3. Isotope-effect studies with NADH and its deuterium analogue show that the rapidly formed ternary complex with o-nitrophenylpyruvate slowly isomerizes to give an active ternary complex, which then rapidly decomposes to NAD(+). The isomerization is pH-independent, and it is suggested that histidine-195 is still protonated in the activated ternary complex, which is present before hydride transfer. 4. All four subunits of the enzyme are kinetically equivalent with respect to the oxidation of bound NADH by o-nitrophenylpyruvate. 5. A partial mechanism for the enzyme is described which emphasizes the isomerizations and ionizations involved in forming the reduced ternary complex at pH6 and 8.     

State and accessibility of zinic in yeast alcohol dehydrogenase.

1. Yeast alcohol dehydrogenase (EC 1.1.1.1) is inhibited in the presence of 1,10-phenanthroline. 2. A conformational change in the enzyme's structure is induced by 1,10-phenanthroline, and is abolished in the presence of NADH. 1,10-Phenanthroline binds to the enzyme competitively with respect to NADH, with a stoicheiometry of 2 mol of 1,10-phenanthroline/144000g of enzyme. 3. 1,10-Phenanthroline induces a time-dependent dissociation of Zn2+ from the enzyme, which is in correlation with its inhibitions. 4. Spectrophotometric measurement indicates that the dissociation of half (2 zinc atoms/tetramer) of the total zinc content of the enzyme correlates with the full inhibition of its activity. Measurement of the tightly bound Zn2+ by atomic absorption photometry confirms this. 5. A proposition is advanced that the tetrameric molecule of yeast alcohol dehydrogenase possesses an inherent asymmetry, with four monomeric subunits being arranged in two mutually symmetrical pairs.     

The role of nicotinamide adenine dinucleotide in the inhibition of bovine liver S-adenosylhomocysteine hydrolase by neplanocin A

Neoplanocin A, a cyclopentenyl analog of adenosine, has been shown recently to be a tight binding inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1), exhibiting a stoichiometry of one molecule of inhibitor per molecule of the enzyme tetramer (Borchardt, R. T., Keller, B. T., and Patel-Thombre, U. (1984) J. Biol. Chem. 259, 4353-4358). In the present study a detailed analysis was performed of the possible role of the enzyme-bound NAD+ in the inactivation of AdoHcy hydrolase by neplanocin A. The NAD+/NADH content was quantitated using a fluorescence technique. The native enzyme showed intrinsic fluorescence with an emission maximum at 460 nm when excited at 340 nm, partially due to NADH bound to the enzyme. It was found that the content of NAD+ and NADH in freshly prepared, native enzyme is equal, having a stoichiometry of two nucleotides per enzyme molecule (tetramer). In addition, it was observed that the enzymatic activity of the native enzyme can be increased by about 30% following preincubation with NAD+. Furthermore, it was demonstrated that the mechanism of inhibition of AdoHcy hydrolase by neplanocin A involves the reduction of enzymatically bound NAD+ to NADH. Catalytic activity of the inactivated enzyme could be fully recovered in a time-dependent manner by further incubation with NAD+ (but not NADH). It was also found that inhibition by neplanocin A does not involve dissociation of the bound NAD+ or NADH from the enzyme, but simply reduction of the NAD+ to NADH.     

Reaction of the 2',3'-dialdehyde derivative of NADPH at a nucleotide site of bovine liver glutamate dehydrogenase

The 2',3'-dialdehyde derivative of NADPH (oNADPH) acts as a coenzyme for the reaction catalyzed by bovine liver glutamate dehydrogenase. Incubation of 250 microM oNADPH with enzyme for 300 min at 30 degrees C and pH 8.0 yields covalent incorporation of 1.0 mol of oNADPH/mol of enzyme subunit. The modified enzyme has a functional catalytic site and is activated by ADP, but is no longer inhibited by high NADH concentrations and exhibits decreased sensitivity to GTP inhibition. Using the change in inhibition by 600 microM NADH or 1 microM GTP to monitor the reaction leads to rate constants of 44.0 and 41.5 min-1 M-1, respectively, suggesting that loss of inhibition by the two regulatory compounds results from reaction by oNADPH at a single location. The oNADPH incorporation is proportional to the decreased inhibition by 600 microM NADH or 1 microM GTP, extrapolating to less than 1 mol of oNADPH/mol of subunit when the maximum change in NADH or GTP inhibition has occurred. Modified enzyme is still 93% inhibited at saturating levels of GTP, although its K1 is increased 20-fold to 4.6 microM. The kinetic effects caused by oNADPH are not prevented by alpha-ketoglutarate, ADP, 5 mM NADH, or 200 microM GTP alone, but are prevented by 5 mM NADH with 200 microM GTP. Incorporation of oNADPH into enzyme at 255 min is 0.94 mol/mol of peptide chain in the absence of ligands but only 0.53 mol/mol of peptide chain in the presence of the protectants 5 mM NADH plus 200 microM GTP. These results indicate that oNADPH modifies specifically about 0.4-0.5 sites/enzyme subunit or about 3 sites/enzyme hexamer and that reaction occurs at a GTP-dependent inhibitory NADH site of glutamate dehydrogenase.     

Deoxyhypusine synthase generates and uses bound NADH in a transient hydride transfer mechanism

Deoxyhypusine is a modified lysine residue. It is formed posttranslationally in the precursor of eukaryotic initiation factor 5A (eIF5A) by deoxyhypusine synthase, employing spermidine as a butylamine donor. In the initial step of this reaction, deoxyhypusine synthase catalyzes the production of NADH through dehydrogenation of spermidine. Fluorescence measurements of this reaction revealed a -22-nm blue shift in the emission peak of NADH and a approximately 15-fold increase in peak intensity, characteristics of tightly bound NADH that were not seen by simply mixing NADH and enzyme. The fluorescent properties of the bound NADH can be ascribed to a hydrophobic environment and a rigidly held, open conformation of NADH, features in accord with the known crystal structure of the enzyme. Considerable fluorescence resonance energy transfer from tryptophan 327 in the active site to the dihydronicotinamide ring of NADH was seen. Upon addition of the eIF5A precursor, utilization of the enzyme-bound NADH for reduction of the eIF5A-imine intermediate to deoxyhypusine was reflected by a rapid decrease in the NADH fluorescence, indicating a transient hydride transfer mechanism as an integral part of the reaction. The number of NADH molecules bound approached four/enzyme tetramer; not all of the bound NADH was available for reduction of the eIF5A-imine intermediate.     

Some properties of aldehyde dehydrogenase from sheep liver mitochondria.

Aldehyde dehydrogenase from sheep liver mitochondria was purified to homogeneity as judged by electrophoresis on polyacrylamide gels, and by sedimentation-equilibrium experiments in the analytical ultracentrifuge. The enzyme has a molecular weight of 198000 and a subunit size of 48000, indicating that the molecule is a tetramer. Fluorescence and spectrophotometric titrations indicate that each subunit can bind 1 molecule of NADH. Enzymic activity is completely blocked by reaction of 4mol of 5,5'-dithiobis-(2-nitrobenzoate)/mol of enzyme. Excess of disulfiram or iodoacetamide decreases activity to only 50% of the control value, and only two thiol groups per molecule are apparently modified by these reagents.     

Vit C.Fe(III) induced loss of the covalently bound phosphate and enzyme activity of phosphoglucomutase

Rabbit muscle phosphoglucomutase was irreversibly inactivated upon preincubation with vitamin C (Vit C). Fe(III), NADH.NADH oxidase.Fe(III), or ferritin.Vit C. Substrate, glucose 1-phosphate and Mg2+ afforded partial protection. No altered amino acid could be detected in the inactive enzyme. Enzyme so inactivated was more susceptible to trypsin. More importantly, during inactivation, the enzyme lost up to 70% of its enzyme-bound phosphate; the completely inactivated enzyme retained the remainder of the bound phosphate which was isolatable as phosphoserine residing in the 22-amino acid long tryptic peptide. Free phosphoserine as well as those in phosphorylase alpha and phosphocasein were resistant to the oxidizing system, suggesting that the phosphoserine of phosphoglucomutase is uniquely vulnerable to these treatments. Alternatively, a fraction of the total 1 mol of phosphate in the phosphoform of phosphoglucomutase may not be associated with phosphoserine. Phosphoglyceromutase, which has phosphohistidine at its active site, was also inactivated by the oxidizing system. However, it did not release any of the bound phosphate.     

Modification of NAD-dependent isocitrate dehydrogenase by the 2',3'-dialdehyde derivatives of NAD, NADH, NADP, and NADPH

The 2',3'-dialdehyde nicotinamide ribose derivatives of NAD (oNAD) and NADH (oNADH) have been prepared enzymatically from the corresponding 2',3'-dialdehyde analogs of NADP and NADPH. Pig heart NAD-dependent isocitrate dehydrogenase requires NAD as coenzyme but binds NADPH, as well as NADH, ADP, and ATP, at regulatory sites. Incubation of 1-3 mM oNAD or oNADH with this isocitrate dehydrogenase causes a time-dependent decrease in activity to a limiting value 40% that of the initial enzyme, suggesting that reaction does not occur at the catalytic coenzyme site. Upon varying the concentration of oNAD or oNADH from 0.2 to 3 mM, the inactivation rate constants increase in a nonlinear manner, consistent with reversible binding of oNAD and oNADH to the enzyme prior to covalent reaction. Inactivation is accompanied by incorporation of radioactive reagent with extrapolation to 0.54 mol [14C]oNAD or 0.45 mol [14C]oNADH/mol average enzyme subunit (or about 2 mol reagent/mol enzyme tetramer) when the enzyme is maximally inactivated; this value corresponds to the number of reversible binding sites for each of the natural ligands of isocitrate dehydrogenase. The protection against oNAD or oNADH inactivation by NADH, NADPH, and ADP (but not by isocitrate, NAD, or NADP) indicates that reaction occurs in the region of a nucleotide regulatory site. In contrast to the effects of oNAD and oNADH, oNADP and oNADPH cause total inactivation of the NAD-dependent isocitrate dehydrogenase, concomitant with incorporation, respectively, of about 3.5 mol [14C]oNADP or 1.3 mol [14C]oNADPH/mol average subunit. Reaction rates exhibit a linear dependence on [oNADP] or [oNADPH] and protection by natural ligands against inactivation is not striking. These results imply that oNADP and oNADPH are acting in this case as general chemical modifiers and indicate the importance of the free adenosine 2'-OH of oNAD and oNADH for specific labeling of the NAD-dependent isocitrate dehydrogenase. The new availability of 2',3'-dialdehyde nicotinamide ribose derivatives of NAD, NADH, NADP, and NADPH may allow selection of the appropriate reactive coenzyme analog for affinity labeling of a variety of dehydrogenases.