A study of the kinetics and mechanism of yeast alcohol dehydrogenase with a variety of substrates

1. The kinetics of oxidation of ethanol, propan-1-ol, butan-1-ol and propan-2-ol by NAD(+) and of reduction of acetaldehyde and butyraldehyde by NADH catalysed by yeast alcohol dehydrogenase were studied. 2. Results for the aldehyde-NADH reactions are consistent with a compulsory-order mechanism with the rate-limiting step being the dissociation of the product enzyme-NAD(+) complex. In contrast the results for the alcohol-NAD(+) reactions indicate that some dissociation of coenzyme from the active enzyme-NAD(+)-alcohol ternary complexes must occur and that the mechanism is not strictly compulsory-order. The rate-limiting step in ethanol oxidation is the dissociation of the product enzyme-NADH complex but with the other alcohols it is probably the catalytic interconversion of ternary complexes. 3. The rate constants describing the combination of NAD(+) and NADH with the enzyme and the dissociations of these coenzymes from binary complexes with the enzyme were measured.     

Kinetic mechanism from steady-state kinetics of the reaction catalysed by baker''s-yeast glucose 6-phosphate dehydrogenase in solution and covalently attached to sepharose.

1. The reaction catalysed by glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast was studied in 42mM-glycylglycine buffer, pH7.4 at 25 degrees C, by initial-velocity studies and by the use of NADPH as a product inhibitor. 2. The reactions catalysed by both the soluble enzyme and a stable enzyme covalently attached to CNBr-activated Sepharose 4B probably follow an ordered reaction mechanism with NADP+ and NADPH as the leading reactants. 3. The kinetic constants obtained for the soluble enzyme lere: KNADP+m, 19 muM; KNADP+s, 23 muM; KNADPHs, 15 muM. Similar values were obtained for the immobilized enzyme. 4. The assay of the immobilized enzyme was done by using a micro packed-bed recirculation reactor, and the advantages of this technique are discussed.     

Interaction of NADP(H) with oxidized and reduced P450 reductase during catalysis. Studies with nucleotide analogues

Previous studies have shown that the interaction of P450 reductase with bound NADP(H) is essential to ensure fast electron transfer through the two flavin cofactors. In this study we investigated in detail the interaction of the house fly flavoprotein with NADP(H) and a number of nucleotide analogues. 1,4,5,6-Tetrahydro-NADP, an analogue of NADPH, was used to characterize the interaction of P450 reductase with the reduced nucleotide. This analogue is inactive as electron donor, but its binding affinity and rate constant of release are very close to those for NADPH. The 2'-phosphate contributes about 5 kcal/mol of the binding energy of NADP(H). Oxidized nicotinamide does not interact with the oxidized flavoprotein, while reduced nicotinamide contributes 1.3 kcal/mol of the binding energy. Oxidized P450 reductase binds NADPH with a K(d) of 0.3 microM, while the affinity of the reduced enzyme is considerably lower, K(d) = 1.9 microM. P450 reductase catalyzes a transhydrogenase reaction between NADPH and oxidized nucleotides, such as thionicotinamide-NADP(+), acetylpyridine-NADP(+), or [(3)H]NADP(+). The reverse reaction, reduction of [(3)H]NADP(+) by the reduced analogues, is also catalyzed by P450 reductase. We define the mechanism of the transhydrogenase reaction as follows: NADPH binding, hydride ion transfer, and release of the NADP(+) formed. An NADP(+) or its analogue binds to the two-electron-reduced flavoprotein, and the electron-transfer steps reverse to transfer hydride ion to the oxidized nucleotide, which is released. Measurements of the flavin semiquinone content, rate constant for NADPH release, and transhydrogenase turnover rates allowed us to estimate the steady-state distribution of P450 reductase species during catalysis, and to calculate equilibrium constants for the interconversion of catalytic intermediates. Our results demonstrate that equilibrium redox potentials of the flavin cofactors are not the sole factor governing rapid electron transfer during catalysis, but conformational changes must be considered to understand P450 reductase catalysis.     

Chemical mechanism of lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung. pH-dependence of kinetic parameters.

Lysophosphatidylcholine: lysophosphatidylcholine acyltransferase is an enzyme that catalyses two reactions: hydrolysis of lysophosphatidylcholine and transacylation between two molecules of lysophosphatidylcholine to give disaturated phosphatidylcholine. Following the kinetic model previously proposed for this enzyme [Martín, Pérez-Gil, Acebal & Arche (1990) Biochem. J. 266, 47-53], the values of essential pK values in free enzyme and substrate-enzyme complexes have now been determined. The chemical mechanism of catalysis was dependent on the deprotonation of a histidine residue with pK about 5.7. This result was supported by the perturbation of pK values by addition of organic solvent. Very high and exothermic enthalpy of ionization was measured, indicating that a conformational re-arrangement in the enzyme accompanies the ionization of the essential histidine residue. These results, as well as the results from previous studies, enabled the proposal of a chemical mechanism for the enzymic reactions catalysed by lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung.     

Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii

A method is described for the partial purification of pyridine nucleotide transhydrogenase from Azotobacter vinelandii (ATCC 9104) cells. The most highly purified preparation catalyzes the reduction of 300 mumoles of nicotinamide adenine dinucleotide (NAD(+)) per min per mg of protein under the assay conditions employed. The enzyme catalyzes the reduction of NAD(+), deamino-NAD(+), and thio-NAD(+) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as hydrogen donor, and the reduction of nicotinamide adenine dinucleotide phosphate (NADP(+)) and thio-NAD(+) with reduced NAD (NADH) as hydrogen donor. The reduction of acetylpyridine AD(+), pyridinealdehyde AD(+), acetylpyridine deamino AD(+), and pyridinealdehydedeamino AD(+) with NADPH as hydrogen donor was not catalyzed. The enzyme catalyzes the transfer of hydrogen more readily from NADPH than from NADH with different hydrogen acceptors. The transfer of hydrogen from NADH to NADP(+) and thio-NAD(+) was markedly stimulated by 2'-adenosine monophosphate (2'-AMP) and inhibited by adenosine diphosphate (ADP), adenosine triphosphate (ATP), and phosphate ions. The transfer of hydrogen from NADPH to NAD(+) was only slightly affected by phosphate ions and 2'-AMP, except at very high concentrations of the latter reagent. In addition, the transfer of hydrogen from NADPH to thio-NAD(+) was only slightly influenced by 2'-AMP, ADP, ATP, and other nucleotides. The kinetics of the transhydrogenase reactions which utilized thio-NAD(+) as hydrogen acceptor and NADH or NADPH as hydrogen donor were studied in some detail. The results suggest that there are distinct binding sites for NADH and NAD(+) and perhaps a third regulator site for NADP(+) or 2'-AMP. The heats of activation for the transhydrogenase reactions were determined. The properties of this enzyme are compared with those of other partially purified transhydrogenases with respect to the regulatory functions of 2'-AMP and other nucleotides on the direction of flow of hydrogen between NAD(+) and NADP(+).     

The nicotinamide nucleotide specificity of glutamate dehydrogenase in intact RAT-liver mitochondria

1. The kinetics of oxidation of intramitochondrial reduced nicotinamide nucleotides by -oxoglutarate plus ammonia in intact rat-liver mitochondria have been reinvestigated. It is demonstrated that the preferential oxidation of NADPH observed on addition of ammonia to mitochondria, preincubated under energized conditions in the presence of -oxoglutarate, is due to a transhydrogenation catalysed by glutamate dehydrogenase rather than to an energy-dependent modification of the nicotinamide nucleotide specificity of the enzyme in intact mitochondria.

2. When mitochondria are preincubated at 25 °C under energized conditions in the presence of respiratory inhibitors with the substrates of glutamate dehydrogenase, an oxidation of NADPH, but not of NADH, is brought about by decreasing the reaction temperature. Both the rate of NADPH oxidation and the final steady-state mass-action ratio of nicotinamide nucleotides are dependent on the concentration of ammonia and on the final reaction temperature. A similar effect is observed when rhein is added to the reaction medium at 25 °C in order to inhibit the energy-linked transhydrogenase reaction.

3. In the presence of the substrates of glutamate dehydrogenase, intact ratliver mitochondria catalyse an ATPase reaction due to the simultaneous activity of the energy-linked transhydrogenase and the non-energy-linked transhydrogenation catalysed by glutamate dehydrogenase.

4. These findings are discussed in relation to the nicotinamide nucleotide specificity of glutamate dehydrogenase and to a possible compartmentation of nicotinamide nucleotides in intact rat-liver mitochondria.     

NADP(+)-dependent D-threonine dehydrogenase from Pseudomonas cruciviae IFO 12047.

NADP(+)-dependent D-threonine dehydrogenase (EC 1.1.1.-), which catalyzes the oxidation of the 3-hydroxyl group of D-threonine, was purified to homogeneity from a crude extract of Pseudomonas cruciviae IFO 12047. The enzyme had a molecular mass of about 60,000 Da and consisted of two identical subunits. In addition to D-threonine, D-threo-3-phenylserine, D-threo-3-thienylserine, and D-threo-3-hydroxynorvaline were also substrates. However, the other isomers of threonine and 3-phenylserine were inert. The enzyme showed maximal activity at pH 10.5 for the oxidation of D-threonine. The enzyme required NADP+. NAD+ showed only slight activity. The enzyme was not inhibited by EDTA, o-phenanthroline, alpha,alpha'-dipyridyl, HgCl2, or p-chloromercuribenzoate but was inhibited by tartronate, malonate, pyruvate, and DL-2-hydroxybutyrate. The inhibition by these organic acids was competitive against D-threonine. Initial-velocity and product inhibition studies suggested that the oxidation proceeded through a sequential ordered Bi Bi mechanism. The Michaelis constants for D-threonine and NADP+ were 13 and 0.12 mM, respectively.     

Regulation of poly-β-hydroxybutyrate biosynthesis by nicotinamide nucleotide in Alcaligenes eutrophus

Abstract Poly-β-hydroxybutyrate biosynthesis was studied in Alcaligenes eutrophus under various nutrient-limiting conditions. When the cells were cultivated in nitrogen-limited media, both the levels of NAD(P)H and the ratios of NAD(P)H/NAD(P) were higher than those under nitrogen-sufficient conditions. The specific poly-β-hydroxybutyrate production rate was found to increase with the values of both NADH/NAD and NADPH/NADP, indicating that poly-β-hydroxybutyrate synthesis is directly regulated by the ratios of nicotinamide nucleotides. The effects of nicotinamide nucleotides on poly-β-hydroxybutyrate biosynthesis was investigated with regard to enzyme kinetics. Citrate synthase activity was significantly inhibited by NADH and NADPH, indicating that poly-β-hydroxybutyrate accumulation could be enhanced by facilitating the metabolic flux of acetyl-CoA to poly-β-hydroxybutyrate synthetic pathway. It was also found that cellular NADPH was a limiting substrate for NADPH-linked reductase, controlling the overall biosynthetic activity of poly-/3-hydroxybutyrate in this strain.     

A common active site model for catalysis by chorismate mutase--prephenate dehydrogenase.

Comparison of the calculated structures for the transition states of the two reactions catalysed by chorismate mutase prephenate dehydrogenase suggests that both reactions could be catalysed at a common active site. Kinetic data for the enzyme from Aerobacteraerogenes are consistent with this possibility. On the basis of these theoretical and experimental data a model for a common active site is developed. In the model, the transition state for each reaction is bound to the enzyme via both of the two substrate carboxyl groups, and can also interact with the coenzyme nicotinamide adenine dinucleotide through a hydrogen bond between the amide moiety of the nicotinamide ring and the hydroxyl group of the substrate. Chorismate, prephenate and 4-hydroxyphenylpyruvate in their ground states form the same hydrogen bond to the coenzyme, but are bound to the enzyme via a single carboxyl group only. The additional bond formed between the enzyme and the transition state structures thus provides the transition state stabilization required for catalysis of both reactions.     

Insights into the mechanism of bovine CD38/NAD+glycohydrolase from the X-ray structures of its michaelis complex and covalently-trapped intermediates

Bovine CD38/NAD(+)glycohydrolase (bCD38) catalyses the hydrolysis of NAD(+) into nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose (cADPR). We solved the crystal structures of the mono N-glycosylated forms of the ecto-domain of bCD38 or the catalytic residue mutant Glu218Gln in their apo state or bound to aFNAD or rFNAD, two 2'-fluorinated analogs of NAD(+). Both compounds behave as mechanism-based inhibitors, allowing the trapping of a reaction intermediate covalently linked to Glu218. Compared to the non-covalent (Michaelis) complex, the ligands adopt a more folded conformation in the covalent complexes. Altogether these crystallographic snapshots along the reaction pathway reveal the drastic conformational rearrangements undergone by the ligand during catalysis with the repositioning of its adenine ring from a solvent-exposed position stacked against Trp168 to a more buried position stacked against Trp181. This adenine flipping between conserved tryptophans is a prerequisite for the proper positioning of the N1 of the adenine ring to perform the nucleophilic attack on the C1' of the ribofuranoside ring ultimately yielding cADPR. In all structures, however, the adenine ring adopts the most thermodynamically favorable anti conformation, explaining why cyclization, which requires a syn conformation, remains a rare alternate event in the reactions catalyzed by bCD38 (cADPR represents only 1% of the reaction products). In the Michaelis complex, the substrate is bound in a constrained conformation; the enzyme uses this ground-state destabilization, in addition to a hydrophobic environment and desolvation of the nicotinamide-ribosyl bond, to destabilize the scissile bond leading to the formation of a ribooxocarbenium ion intermediate. The Glu218 side chain stabilizes this reaction intermediate and plays another important role during catalysis by polarizing the 2'-OH of the substrate NAD(+). Based on our structural analysis and data on active site mutants, we propose a detailed analysis of the catalytic mechanism.     

Structures of the dI2dIII1 complex of proton-translocating transhydrogenase with bound, inactive analogues of NADH and NADPH reveal active site geometries

Transhydrogenase couples the redox reaction between NADH and NADP+ to proton translocation across a membrane. The enzyme comprises three components; dI binds NAD(H), dIII binds NADP(H), and dII spans the membrane. The 1,4,5,6-tetrahydro analogue of NADH (designated H2NADH) bound to isolated dI from Rhodospirillum rubrum transhydrogenase with similar affinity to the physiological nucleotide. Binding of either NADH or H2NADH led to closure of the dI mobile loop. The 1,4,5,6-tetrahydro analogue of NADPH (H2NADPH) bound very tightly to isolated R. rubrum dIII, but the rate constant for dissociation was greater than that for NADPH. The replacement of NADP+ on dIII either with H2NADPH or with NADPH caused a similar set of chemical shift alterations, signifying an equivalent conformational change. Despite similar binding properties to the natural nucleotides, neither H2NADH nor H2NADPH could serve as a hydride donor in transhydrogenation reactions. Mixtures of dI and dIII form dI2dIII1 complexes. The nucleotide charge distribution of complexes loaded either with H2NADH and NADP+ or with NAD+ and H2NADPH should more closely mimic the ground states for forward and reverse hydride transfer, respectively, than previously studied dead-end species. Crystal structures of such complexes at 2.6 and 2.3 A resolution are described. A transition state for hydride transfer between dihydronicotinamide and nicotinamide derivatives determined in ab initio quantum mechanical calculations resembles the organization of nucleotides in the transhydrogenase active site in the crystal structure. Molecular dynamics simulations of the enzyme indicate that the (dihydro)nicotinamide rings remain close to a ground state for hydride transfer throughout a 1.4 ns trajectory.     

Transient-phase kinetic studies on the nucleotide binding to 3alpha-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 using fluorescence stopped-flow procedures.

The dual nucleotide cofactor-specific enzyme, 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) from Pseudomonas sp. B-0831, is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. Transient-phase kinetic studies using the fluorescence stopped-flow method were conducted with 3alpha-HSD to characterize the nucleotide binding mechanism. The binding of oxidized nucleotides, NAD(+), NADP(+) and nicotinic acid adenine dinucleotide (NAAD(+)), agreed well with a one-step mechanism, while that of reduced nucleotide, NADH, showed a two-step mechanism. This difference draws attention to previous characteristic findings on rat liver 3alpha-HSD, which is a member of the aldo-keto reductase (AKR) superfamily. Although functionally similar, AKRs are structurally different from SDRs. The dissociation rate constants associated with the enzyme-nucleotide complex formation were larger than the k(cat) values for either oxidation or reduction of substrates, indicating that the release of cofactors is not rate-limiting overall. It should also be noted that k(cat) for a substrate, cholic acid, with NADP(+) was only 6% of that with NAD(+), and no catalytic activity was detectable with NAAD(+), despite the similar binding affinities of nucleotides. These results suggest that a certain type of nucleotide can modulate nucleotide-binding mode and further the catalytic function of the enzyme.     

Purification and properties of the H(+)-nicotinamide nucleotide transhydrogenase from Rhodobacter capsulatus.

1. H(+)-transhydrogenase from Rhodobacter capsulatus is an integral membrane protein which, unlike the enzyme from Rhodospirillum rubrum, does not require the presence of a water-soluble component for activity. 2. The enzyme from Rb. capsulatus was solubilised in Triton X-100 and subjected to ion-exchange, hydroxyapatite and then gel-exclusion column chromatography. SDS/PAGE of the purified enzyme revealed the presence of two polypeptides with apparent Mr 53,000 and 48,000. Other minor components which were stained on the electrophoresis gels or which were revealed on Western blots exposed to antibodies raised to total membrane proteins, were probably contaminants. 3. Antibodies raised to the 53-kDa and 48-kDa polypeptides cross-reacted with equivalent polypeptides in Western blots of solubilised membranes from Rb. capsulatus, Rhodobacter sphaeroides and Rhs. rubrum. The significance of this finding is discussed in the context of the hypothesis [Fisher, R.R. & Earle, S.R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York] that the soluble component associated with H(+)-transhydrogenase from Rhs. rubrum is an integral part of the catalytic machinery. Antibodies against the 48-kDa and 53-kDa polypeptides of the Rb. capsulatus enzyme cross-reacted with equivalent polypeptides in solubilised membranes of Escherichia coli. 4. The dependence of the rate of H- transfer by purified H(+)-transhydrogenase on the nucleotide substrate concentrations under steady-state conditions, the effects of inhibition by nucleotide products and the inhibition by 2'-AMP and by 5'-AMP suggest that the reaction proceeds by the random addition of substrates to the enzyme with the formation of a ternary complex. 5. In conflict with this conclusion, the reduction of acetylpyridine adenine dinucleotide (AcPdAD+) by NADH in the absence of NADP+ by bacterial membranes was earlier taken as evidence for the existence of a reduced enzyme intermediate [Fisher, R.R. & Earle, S.R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York]. However, it is shown here that although chromatophore membranes of Rb. capsulatus catalysed the reduction of AcPdAD+ by NADH, the reaction was not associated with the purified H(+)-transhydrogenase. Moreover, in contrast with the true transhydrogenase reaction, the reconstitution of AcPdAD+ reduction by NADH (in the absence of NADP+) in washed membranes of Rhs. rubrum with partially purified transhydrogenase factor, was only additive.     

Mechanism of non-enzymic transamination reaction between histidine and α-oxoglutaric acid

Non-enzymic transamination reactions at 85 degrees between various amino acids and alpha-oxoglutaric acid are catalysed by metal ions, e.g. Al(3+), Fe(2+), Cu(2+) and Fe(3+). The reaction is optimum at pH4.0. Of the 14 amino acids studied histidine is the most active. In the presence of Al(3+) histidine transaminates with alpha-oxoglutaric acid, forming glutamic acid and Al(3+)-imidazolylpyruvic acid complex as the end products. However, in the presence of Fe(2+) or Cu(2+) the products are glutamic acid and a 1:2 metal ion-imidazolylpyruvic acid chelate. The greater effectiveness of histidine in these reactions is attributed to the presence of the tertiary imidazole nitrogen atom, which is involved in the formation of stable sparingly soluble metal ion-imidazolylpyruvic acid complexes or chelates as end products of these reactions. Of the metal ions studied only Al(3+), Fe(2+), Fe(3+) and Cu(2+) are effective catalysts for the transamination reactions, and EDTA addition completely inhibits the catalytic effect of the Al(3+). Spectrophotometric evidence is presented to demonstrate the presence of metal ion complexes of Schiff bases of histidine as intermediates in the transamination reactions. These results may contribute to understanding the role of histidine in enzyme catalysis.     

Novel psychrophilic and thermolabile L-threonine dehydrogenase from psychrophilic Cytophaga sp. strain KUC-1

A psychrophilic bacterium, Cytophaga sp. strain KUC-1, that abundantly produces a NAD(+)-dependent L-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the Cytophaga enzyme is distinct from other L-threonine dehydrogenases that have thus far been studied. L-Threonine and DL-threo-3-hydroxynorvaline were the substrates, and NAD(+) and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45 degrees C. The kinetic parameters of the enzyme are highly influenced by temperatures. The K(m) for L-threonine was lowest at 20 degrees C. Dead-end inhibition studies with pyruvate and adenosine-5'-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD(+) binds to an enzyme prior to L-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 bp encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast, L-threonine dehydrogenase from E. coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme. L-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the E. coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the substrate binding.     

D-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi: molecular cloning of the enzyme gene and crystal structure of the enzyme

The gene coding for d-3-hydroxybutyrate dehydrogenase (HBDH) was cloned from Pseudomonas fragi. The nucleotide sequence contained a 780 bp open reading frame encoding a 260 amino acid residue protein. The recombinant enzyme was efficiently expressed in Escherichia coli cells harboring pHBDH11 and was purified to homogeneity as judged by SDS-PAGE. The enzyme showed a strict stereospecificity to the D-enantiomer (3R-configuration) of 3-hydroxybutyrate as a substrate. Crystals of the ligand-free HBDH and of the enzyme-NAD+ complex were obtained using the hanging-drop, vapor-diffusion method. The crystal structure of the HBDH was solved by the multiwavelength anomalous diffraction method using the SeMet-substituted enzyme and was refined to 2.0 A resolution. The overall structure of P.fragi HBDH, including the catalytic tetrad of Asn114, Ser142, Tyr155, and Lys159, shows obvious relationships with other members of the short-chain dehydrogenase/reductase (SDR) family. A cacodylate anion was observed in both the ligand-free enzyme and the enzyme-NAD+ complex, and was located near the catalytic tetrad. It was shown that the cacodylate inhibited the NAD+-dependent D-3-hydroxybutyrate dehydrogenation competitively, with a Ki value of 5.6 mM. From the interactions between cacodylate and the enzyme, it is predicted that substrate specificity is achieved through the recognition of the 3-methyl and carboxyl groups of the substrate.     

Nicotinamide nucleotides in the erythrocytes of patients suffering from pellagra

1. The nicotinamide nucleotide concentrations in the erythrocytes of subjects suffering from pellagra (pellagrins) were not lower than those in normal subjects, but the ability of erythrocytes to synthesize these nucleotides in vitro was significantly lower in pellagrins. 2. The oral administration of 10g. of l-leucine daily for 5 days depressed the nicotinamide nucleotide-synthesizing ability of erythrocytes both in normal subjects and in pellagrins. This was not accompanied by changes in the nucleotide concentration in erythrocytes. 3. Quinolinic acid brought about a significant inhibition of the synthesis of nicotinamide nucleotides in vitro. Such inhibition was partially overcome by increasing the concentration of nicotinic acid in the medium.     

Kinetic analysis of duck epsilon-crystallin, a lens structural protein with lactate dehydrogenase activity.

Biochemical characterization and kinetic analysis of epsilon-crystallin from the lenses of common ducks were undertaken to elucidate the enzyme mechanism of this unique crystallin with lactate dehydrogenase (LDH) activity. Despite the structural similarities between epsilon-crystallin and chicken heart LDH, differences in charge and kinetic properties were revealed by isoenzyme electrophoresis and kinetic studies. Bi-substrate kinetic analysis examined by initial-velocity and product-inhibition studies suggested a compulsory ordered Bi Bi sequential mechanism with NADH as the leading substrate followed by pyruvate. The products were released in the order L-lactate and NAD+. The catalysed reaction is shown to have a higher rate in the formation of L-lactate and NAD+. Substrate inhibition was observed at high concentrations of pyruvate and L-lactate for the forward and reverse reactions respectively. The substrate inhibition was presumably due to the formation of epsilon-crystallin-NAD(+)-pyruvate or epsilon-crystallin-NADH-L-lactate abortive ternary complexes, as suggested by the product-inhibition studies. The significance and the interrelationship of duck epsilon-crystallin with other well-known LDHs are discussed with special regard to its role as a structural protein with some enzymic function in lens metabolism.