The enthalpy of protolysis of liver alcohol dehydrogenase upon binding nicotinamide adenine dinucleotide.

The binding of NAD+, NADH, and ADP-ribose to horse liver alcohol dehydrogenase has been studied calorimetrically as a function of pH at 25 degrees C. The enthalpy of NADH binding is 0 +/- 0.5 kcal mol-1 in the pH range 6 to 8.6. The enthalpy of NAD+ binding, however, varies with pH in a sigmoidal fashion and is -4.0 kcal mol(NAD)-1 at pH 6.0 and +4.5 kcal mol(NAD)-1 at pH 8.6 with an apparent pKa of 7.6 +/- 0.2. The enthalpy of proton ionization of the group on the enzyme is calculated to be in the range 8.8 to 9.8 kcal mol(H+)-1. In conjunction with the available thermodynamic data on the ionization of zinc-bound water in model compounds, it is concluded that the group with a pKa of 9.8 in the free enzyme and 7.6 in the enzyme . NAD+ binary complex is, most likely, the zinc-bound water molecule. Our studies with zinc-free enzyme provide further evidence for this conclusion. Therefore, the processes involving a conformational change of the enzyme upon NAD+ binding and the suggested mechanism of subsequent quenching of the fluorescence of Trp-314 implicating the participation of an ionized tyrosine group must be re-evaluated in the light of this thermodynamic study.     

Effect of pH on coenzyme binding to liver alcohol dehydrogenase.

1. The transient-state kinetics of ligand-displacement reactions have been analyzed. Methods based on this analysis have been used to obtain reliable estimates of on-velocity and off-velocity constants for coenzyme binding to liver alcohol dehydrogenase at different pH values between 6 and 10. 2. The rate of NADH dissociation from the enzyme shows no pronounced dependence on pH. The rate of NAD+ dissociation is controlled by a group with a pKa of 7.6, agreeing with the pKa reported to regulate the binding of certain inhibitory substrate analogues to the enzyme . NAD+ complex. 3. Critical experiments have been performed to test a recent proposal that on-velocity constants for the binding of NADH and NAD+ are controlled by proton equilibria exhibiting different pKa values. The results show that association rates for NADH and NAD+ exhibit the same pH dependence corresponding to a pKa of 9.2. Titrimetric evidence is presented indicating that the latter effect of pH derives from ionization of a group which affects the anion-binding capacity of the coenzyme-binding site.     

Equilibrium binding of nicotinamide nucleotides to lactate dehydrogenases

1. No discontinuities were observed during the continuous titration with NADH of the lactate dehydrogenases of ox muscle, pig heart, pig muscle, rabbit muscle, dogfish muscle or lobster tail muscle. The binding was monitored by either the enhanced fluorescence of bound NADH or the quenched fluorescence of the protein. A single macroscopic dissociation constant, independent of protein concentration, could be used to describe the binding to each enzyme, and there was no need to postulate the involvement of molecular relaxation effects. 2. The affinity for NADH decreases only threefold between pH6 and 8.5. Above pH9 the affinity decreases more rapidly with increasing pH and is consistent with a group of about pK9.5 facilitating binding. Muscle enzymes bind NADH more weakly than does the pig heart enzyme. 3. Increasing temperature and increasing concentrations of ethanol both weaken NADH binding. 4. NADH binding is weakened by increasing ionic strength. NaCl is more effective than similar ionic strengths derived from sodium phosphate or sodium pyrophosphate. 5. Commercial NAD(+) quenches the protein fluorescence of the heart and muscle isoenzymes. Highly purified NAD(+) does not, and its binding was monitored by competition for the NADH-binding sites. A single macroscopic dissociation constant is sufficient to describe NAD(+) binding at the concentrations tested. The dissociation constant is about 0.3mm and is not sensitive to changed ionic strength and to changed pH in the range pH6-8.5.     

Effect of coenzymes on the quaternary structure conformation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Kinetics of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle have been studied under different pH conditions in the absence and presence of various concentrations of NAD+ and NADH. The data have been discussed with respect to the effect of the coenzymes on the quaternary structure symmetry of the two enzymes and their binding isotherms. Both the (homo-tetrameric) apo-enzymes exhibit biphasic kinetics of thermal inactivation, characteristic of C2 symmetry, at lower pH values and a single exponential decay of enzyme activity, characteristic of D2 symmetry, at higher pHs. In each case, NAD+ has no effect on the biphasic kinetic pattern of thermal inactivation at lower pH values, but NADH brings about a change to single exponential decay. At higher pH values, NADH does not affect the kinetic pattern (single exponential decay) of any enzyme, but NAD+ alters it to biphasic kinetics in each case. The data suggest that NAD+ and NADH have higher affinity for the C2 and D2 symmetry conformation, respectively. With mung beans enzyme, the effect of NAD+ on the two rate constants of biphasic inactivation at pH 7.3 is consistent with a Kdiss equal to 110 microM. The NAD(+)-dependent changes in the kinetic pattern of thermal inactivation of this enzyme at pH 8.6 suggest a positive cooperativity in the coenzyme binding (nH = 3.0). In the binding of NADH to the mung beans enzyme, a weak positive cooperativity is observed at pH 7.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Binding of nicotinamide–adenine dinucleotides to diphtheria toxin

1. Changes in protein fluorescence have been utilized in determining the stoicheiometry and dissociation constants of the complexes of diphtheria toxin with NADH(2), NAD, NADPH(2) and NADP. 2. The binding stoicheiometry is 2moles of NADH(2) and 1mole of NADPH(2)/mole of diphtheria toxin. The binding sites for NADH(2) appear to be equivalent and independent. 3. The toxin shows a higher affinity for the reduced than for the oxidized forms of the nucleotides. 4. Dissociation constants at 0.01I, pH7 and 25 degrees are 0.7x10(-6)m for NADH(2) and 0.45x10(-6)m for NADPH(2). Dissociation constants increase with increasing ionic strength, indicating that the binding is mainly electrostatic. 5. Bound NADH(2) and NADPH(2) may be activated to fluoresce by the transfer of energy from the excited aromatic amino acids of the toxin. Activation and emission spectra of bound and free nucleotides are compared. 6. Since NAD and NADH(2) are cofactors specifically required for the inhibition of protein synthesis by diphtheria toxin, the possible role of toxin-nucleotide complexes is discussed in this regard.     

Pyridine nucleotide interaction with rat liver dihydropteridine reductase.

The interactions of a homogeneous preparation of rat liver dihydropteridine reductase with NADH, NADPH, NAD+, NADP+, and the 1-N6-ethenoadenine derivative of NAD+ have been investigated by fluorescence titration, circular dichroism, equilibrium dialysis, Sephadex G-25 chromatography, and polyacrylamide gel electrophoresis. The procedures indicate that the dimeric enzyme has a definite preference for NADH, but binds only 1 mol of this nucleotide per mol of enzyme. The binary complex of enzyme with NADH is only partially stable to exhaustive dialysis and gel electrophoresis, where it shows greater mobility (0.26) than the free enzyme (0.21); however, the complex can be isolated by Sephadex G-25 chromatography, and characterized with respect to its absorbance spectrum. No ternary complexes are observed when samples of reductase, preincubated with excess NADH, and either the reaction product, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, or the inhibitor, methotrexate, are subjected to polyacrylamide gel electrophoresis.     

Relationship between hydroxypyruvate and the production of oxalate in vitro.

Chicken liver lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC1.1.1.27) catalyses the reversible reduction reaction of hydroxypyruvate to L-glycerate. It also catalyses the oxidation reaction of the hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form to glycolate. At pH 8, these latter two reactions are coupled. The coupled system equilibrium is attained when the NAD+/NADH ratio is greater than unity. Hydroxypyruvate binds to the enzyme at the same site as the pyruvate. When there are substances with greater affinity to this site in the reaction medium and their concentration is very high, hydroxypyruvate binds to the enzyme at the L-lactate site. In vitro and with purified preparation of lactate dehydrogenase, hydroxypyruvate stimulates the production of oxalate from glyoxylate-hydrated form and from NAD; the effect is due to the fact that hydroxypyruvate prevents the binding of non-hydrated form of glyoxylate to the lactate dehydrogenase in the pyruvate binding site. At pH 8, THE L-glycerate stimulates the production of glycolate from glyoxylate-non-hydrated form and NADH since hydroxypyruvate prevents the binding of glyoxylate-hydrated form to the enzyme     

UDPglucose dehydrogenase. Kinetics and their mechanistic implications.

Initial velocity and product inhibition studies were carried out on UDP-glucose dehydrogenase (UDPglucose: NAD+ 6-oxidoreductase, EC 1.1.1.22) from beef liver to determine if the kinetics of the reaction are compatible with the established mechanism. An intersecting initial velocity pattern was observed with NAD+ as the variable substrate and UDPG as the changing fixed substrate. UDPglucuronic acid gave competitive inhibition of UDPG and non-competitive inhibition of NAD+. Inhibition by NADH gave complex patterns.Lineweaver-Burk plots of 1/upsilon versus 1/NAD+ at varied levels of NADH gave highly non-linear curves. At levels of NAD+ below 0.05 mM, non-competitive inhibition patterns were observed giving parabolic curves. Extrapolation to saturation with NAD+ showed NADH gave linear uncompetitive inhibition of UDPG if NAD+ was saturating. However, at levels of NAD+ above 0.10 mM, NADH became a competitive inhibitor of NAD+ (parabolic curves) and when NAD+ was saturating NADH gave no inhibition of UDPG. NADH was non-competitive versus UDPG when NAD+ was not saturating. These results are compatible with a mechanism in which UDPG binds first, followed by NAD+, which is reduced and released. A second mol of NAD+ is then bound, reduced, and released. The irreversible step in the reaction must occur after the release of the second mol of NADH but before the release of UDPglucuronic acid. This is apparently caused by the hydrolysis of a thiol ester between UDPglucoronic acid and the essential thiol group of the enzyme. Examination of rate equations indicated that this hydrolysis is the rate-limiting step in the overall reaction. The discontinuity in the velocities observed at high NAD+ concentrations is apparently caused by the binding of NAD+ in the active site after the release of the second mol of NADH, eliminating the NADH inhibition when NAD+ becomes saturating.     

Kinetics of coenzyme binding to liver alcohol dehydrogenase in the pH range 10-12

On- and off-velocity constants for NADH and NAD+ binding to liver alcohol dehydrogenase in the pH range 10-12 have been determined by stopped-flow kinetic methods. The results are consistent with previously reported equilibrium binding data and proposals attributing the main effects of pH on coenzyme binding to ionization of Lys-228 and zinc-bound water. Deprotonation of the group identified as Lys-228 decreases the NADH and NAD+ association rates by a factor exceeding 20 and has no detectable effect on the coenzyme dissociation rates in the examined pH range. Ionization of the group identified as zinc-bound water causes a 3-fold increase of the rate of NADH dissociation from the enzyme, and decreases the rate of NAD+ dissociation by a factor of 200. The NADH and NAD+ association rates are decreased by a factor of 30 and 5, respectively. The observed effects of pH can be rationalized in terms of electrostatic interactions of the ionizing groups with the charges present on the coenzyme molecules and lend support to the idea that binding of the coenzyme nicotinamide ring occurs subsequent to binding of the AMP portion of the coenzyme.     

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.     

Synergism between coenzyme and alcohol binding to liver alcohol dehydrogenase

Heterotropic cooperativity effects in the binding of alcohols and NAD+ or NADH to liver alcohol dehydrogenase have been examined by equilibrium measurements and stopped-flow kinetic studies. Equilibrium data are reported for benzyl alcohol, 2-chloroethanol, 2,2-dichloroethanol, and trifluoroethanol binding to free enzyme over the pH range 6-10. Binary-complex formation between enzyme and alcohols leads to inner-sphere coordination of the alcohol to catalytic zinc and shows a pH dependence reflecting the ionization states of zinc-bound water and the zinc-bound alcohol. The affinity of the binding protonation state of the enzyme for unionized alcohols increases approximately by a factor of 10 on complex formation between enzyme and NAD+ or NADH. The rate and kinetic cooperativity with coenzyme binding of the alcohol association step indicates that enzyme-bound alcohols participate in hydrogen bonding interactions which affect the rates of alcohol and coenzyme equilibration with the enzyme without providing any pronounced contribution to the net energetics of alcohol binding. The pKa values determined for alcohol deprotonation at the binary-complex level are linearly dependent on those of the free alcohols, and can be readily reconciled with the pKa values attributed to ionization of zinc-bound water. Alcohol coordination to catalytic zinc provides a major contribution to the pKa shift which ensures that the substrate is bound predominantly as an alcoholate ion in the catalytically productive ternary complex at physiological pH. The additional pKa shift contributed by NAD+ binding is less pronounced, but may be of particular mechanistic interest since it increases the acidity of zinc-bound alcohols relatively to that of zinc-bound water.     

Specific interactions of 3-phosphoglyceroyl-glyceraldehyde-3-phosphate dehydrogenase with coenzymes.

The binding of oxidized and reduced coenzyme (NAD+ and NADH) to 3-phosphoglyceroyl-glyceraldehyde-3-phosphate dehydrogenase has been studied spectrophotometrically and fluorimetrically. The binding of NAD+ to the acylated sturgeon enzyme is characterized by a significant quenching of the enzyme fluorescence (about 25%) and the induction of a difference spectrum in the ultraviolet absorbance region of the enzyme. Both of these spectroscopic properties are quantitatively distinguishable from those of the corresponding binary enzyme-NAD+ complex. Binding isotherms estimated by gel filtration of the acylated enzyme are in close agreement to those obtained by spectrophotometric and fluorimetric titrations. Up to four NAD+ molecules are bound to the enzyme tetramer. No anticooperativity can be detected in the binding of oxidized coenzyme, which is well described on the basis of a single class of four binding sites with a dissociation constant of 25 muM at 10 degrees C, pH 7.0. The binding of NADH to the acylenzyme has been characterized spectrophotometrically. The absorption band of the dihydronicotinamide moiety of the coenzyme is blue-shifted to 335 nm with respect to free NADH. In addition, a large hypochromicity (23%) is observed together with a significant increase of the bandwidth at half height of this absorption band. This last property is specific to the acylenzyme-DADH complex, since it disappears upon arsenolysis of the acylenzyme. The binding affinity of NADH to the acylated enzyme has been estimated by performing simultaneous spectrophotometric and fluorimetric titrations of the NADH appearance upon addition of NAD+ to a mixture of enzyme and excess glyceraldehyde 3-phosphate. In contrast to NAD+, the reduced coenzyme NADH appears to be relatively strongly bound to the acylated enzyme, the dissociation constant of the acylenzyme-NADH complex being estimated as 2.0 muM at 25 degrees C. In addition a large quenching of the NADH fluorescence (about 83%) is observed. The comparison of the dissociation constants of the coenzyme-acylenzyme complexes and the corresponding Michaelis constants suggests a reaction mechanism of the enzyme in which significant formation and dissociation of NAD+-acylenzyme and NADH-acylenzyme complexes occur. Under physiological conditions the activity of the enzyme can be regulated by the ratio of oxidized and reduced coenzymes. Possible reasons for the lack of anticooperativity in coenzyme binding to the acylated form of the enzyme are discussed.     

Complex formation between nucleotides and D-beta-hydroxybutyrate dehydrogenase studied by fluorescence and EPR spectroscopy

D-beta-Hydroxybutyrate dehydrogenase (D-3-hydroxybutyrate:NAD+ oxidoreductase, EC 1.1.1.30) is a lipid-requiring enzyme which specifically requires phosphosphatidylcholine for enzymic activity. The phosphatidylcholine modifies the binding and orientation of the coenzyme, NAD(H), with respect to the enzyme. In the present study, two derivatives of NAD, spin-labeled either at N-6 or C-8 of the adenine ring, were found to be active as coenzyme. The binding affinity of NADH to the enzyme was opitimized by increasing the salt concentration and increasing the pH from 6 to 8, with the pK at 6.8. Monomethylmalonate, a substrate analogue, was found to enhance NADH binding (Kd is reduced from 4 to 1 microM). Sulfite strongly enhances the binding of NAD+ via the enzyme-catalyzed formation of an adduct of sulfite with the nucleotide; the Kd for binding of NAD-sulfite is in the micromolar range, whereas NAD+ binding is more than a magnitude weaker. The binding of spin-labeled NAD(H) was further characterized by EPR spectroscopy. Increased sensitivity and resolution were obtained with the use of NAD(H) analogues perdeuterated in the spin-label moiety. For these analogues bound to D-beta-hydroxybutyrate dehydrogenase in phospholipid vesicles, EPR studies showed the spin-label moiety to be constrained and revealed two distinct components. Increasing the viscosity of the medium by addition of glycerol affected the EPR spectral characteristics of only the component with the smaller resolved averaged hyperfine splitting. The stage is now set to study motional characteristics of the enzyme, using these spin-labeled probes which mimic the coenzyme.     

The alternative respiratory pathway of the yeast Candida parapsilosis: oxidation of exogenous NAD(P)H

The yeast Candida parapsilosis possesses two routes of electron transfer from exogenous NAD(P)H to oxygen. Electrons are transferred either to the classical cytochrome pathway at the level of ubiquinone through an NAD(P)H dehydrogenase, or to an alternative pathway at the level of cytochrome c through another NAD(P)H dehydrogenase which is insensitive to antimycin A. Analyses of mitoplasts obtained by digitonin/osmotic shock treatment of mitochondria purified on a sucrose gradient indicated that the NADH and NADPH dehydrogenases serving the alternative route were located on the mitochondrial inner membrane. The dehydrogenases could be differentiated by their pH optima and their sensitivity to amytal, butanedione and mersalyl. No transhydrogenase activity occurred between the dehydrogenases, although NADH oxidation was inhibited by NADP+ and butanedione. Studies of the effect of NADP+ on NADH oxidation showed that the NADH:ubiquinone oxidoreductase had Michaelis-Menten kinetics and was inhibited by NADP+, whereas the alternative NADH dehydrogenase had allosteric properties (NADH is a negative effector and is displaced from its regulatory site by NAD+ or NADP+).     

Redox properties of microsomal reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase and cytochrome b5.

Hepatic NADH-cytochrome b5 reductase was reduced by 1 mol of dithionite or NADH per mol of enzyme-bound FAD, without forming a stable semiquinone or intermediate during the titrations. However, the addition of NAD+ to the partially reduced enzyme or illumination in the presence of both NAD+ and EDTA yielded a new intermediate. The intermediate had an absorption band at 375 nm and the optical spectrum resembled anionic semiquinones seen on reduction of other flavin enzymes. Electron paramagnetic resonance measurements confirmed the free-radical nature of the species. To explain the results, a disproportionation reaction between the oxidized and reduced NAD+ complexes (E-FAD-NAD+ + E-FADH2-NAD+ in equilibrium 2E-FADH.-NAD+) is assumed. Potentiometric titration of NADH-cytochrome b5 reductase at pH 7.0 with dithionite gave a midpoint potential of -258 mV; titration with NADH gave -160 mV. This difference may be due to a difference in the relative affinity of NAD+ for the reduced and oxidized forms of the enzyme. The effects of pH on the midpoint potential of the NAD+-free enzyme were very similar to those which have been measured with free FAD. At pH 7.0, midpoint potentials of trypsin-solubilized and detergent-solubilized cytochrome b5 were 13 and 0 mV, respectively.     

Subunit interactions in rabbit-muscle glyceraldehyde-phosphate dehydrogenase, as measured by NAD+ and NADH binding.

1. The binding parameters for NADH and NAD+ to rabbit-muscle glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) have been measured by quenching of the flourescence of the protein and the NADH. 2. The fact that the degree of protein fluorescence quenching by bound NAD+ or NADH, excited at 285 nm and measured at 340 nm ('blue' tryptophans), is not linearly related to the saturation functions of these nucleotides, leads to a slight overestimation of the interaction energy and an underestimation of the concentration of sites, if linearity is assumed. 3. This is also the case for NADH, but not for NAD+, when the protein fluorescence is excited at 305 nm and measured at 390 nm ('red' tryptophans). 4. The binding of NAD+ can be described by a model in which the binding of NAD+, via negative interactions within the dimer, induces weaker binding sites, with the result that the microscopic dissociation constant is 0.08 microM at low saturation and 0.18 microM for the holoenzyme. 5. The binding of NADH can be described on the basis of the same model, the dissociation constant at low saturation being 0.5 microM and of the holoenzyme 1.0 microM. 6. The fluorescence of bound NADH is not sensitive to the conformational changes that cause the decrease in affinity of bound NAD+ or NADH. 7. The binding of NAD+ to the 3-phosphoglyceroyl enzyme can be described by a dissociation constant that is at least two orders of magnitude greater than the dissociation constants of the unacylated enzyme. The affinity of NAD+ to this form of the enzyme is in agreement with the Ki calculated from product inhibition by NAD+ of the reductive dephosphorylation of 1,3-diphosphoglycerate.     

Fast kinetics analysis of the peroxidatic reaction catalysed by horse liver alcohol dehydrogenase

The kinetics of the enzymatic step of the peroxidatic reaction between NAD and hydrogen peroxide, catalysed by horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1), has been investigated at pH 7 at high enzyme concentration. Under such conditions no burst phase has been observed, thus indicating that the rate-limiting step in the process, which converts NAD into Compound I, either precedes or coincides with the chemical step responsible for the observed spectroscopic change. Kinetic analysis of the data, performed according to a simplified reaction scheme suggests that the rate-limiting step is coincident with the spectroscopic (i.e., chemical) step itself. Furthermore, the absence of a proton burst phase indicates the proton release step does not precede the chemical step, in contrast with the case of ethanol oxidation. A kinetic effect of different premixing conditions on the reaction rate has been observed and attributed to the presence of NADH formed in the 'blank reaction' between NAD and residual ethanol tightly bound to alcohol dehydrogenase. A molecular mechanism for the enzymatic peroxidation step is finally proposed, exploiting the knowledge of the much better known reaction of ethanol oxidation. Inhibition of this reaction by NADH has been investigated with respect to H2O2 (noncompetitive, Ki about 10 microM) and to NAD (competitive, Ki about 0.7 microM). The effect of temperature on the steady-state reaction state (about 65 kJ/mol activation energy) has also been studied.     

Characterization of catalytic properties of hydrogenase isolated from the unicellular cyanobacterium Gloeocapsa alpicola CALU 743

The main catalytic properties of the Hox type hydrogenase isolated from the Gloeocapsa alpicola cells have been studied. The enzyme effectively catalyzes reactions of oxidation and evolution of H2 in the presence of methyl viologen (MV) and benzyl viologen (BV). The rates of these reactions in the interaction with the physiological electron donor/acceptor NADH/NAD+ are only 3-8% of the MV(BV)-dependent values. The enzyme interacts with NADP+ and NADPH, but is more specific to NAD+ and NADH. Purification of the hydrogenase was accompanied by destruction of its multimeric structure and the loss of ability to interact with pyridine nucleotides with retained activity of the hydrogenase component (HoxYH). To show the catalytic activity, the enzyme requires reductive activation, which occurs in the presence of H2, and NADH accelerates this process. The final hydrogenase activity depends on the redox potential of the activation medium (E(h)). At pH 7.0, the enzyme activity in the MV-dependent oxidation of H2 increased with a decrease in E(h) from -350 mV and reached the maximum at E(h) of about -390 mV. However, the rate of H2 oxidation in the presence of NAD+ in the E(h) range under study was virtually constant and equal to 7-8% of the maximal rate of H2 oxidation in the presence of MV.     

Affinity chromatography of sn-glycerol-3-phosphate dehydrogenase on immobilized NAD

Three types of potential affinity chromatography columns have been examined for the purification of sn-glycerol-4-phosphate dehydrogenase (EC 1.1.1.8) from rabbit tissues. Each column contained nicotinamide adenine dinucleotide (NAD) covalently attached to an agarose matrix with a different mode of attachment for each column. The most effective column was one in which the NAD was linked to the agarose via the C-8 position of the adenine moiety. Release of the bound enzyme from this column was accomplished by elution with NADH or NAD. The enzymes from brain, heart, kidney, muscle and liver were purified using this procedure with nearly quantitative yields and up to a 90-fold purification. The binding capacity and elution profiles were dependent upon pH, ionic strength and temperature. The capacity was lowest at pH 7 and increased at higher and lower values. Increasing ionic strength and higher temperatures decreased the binding capacities.