Interaction of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase with pyridoxal 5'-diphospho-5'-adenosine. Affinity labeling of Lys-21 and Lys-343

Pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) inhibits glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides competitively with respect to glucose 6-phosphate and noncompetitively with respect to NAD+ or NADP+, with Ki = 40 microM in the NADP-linked and 34 microM in the NAD-linked reaction. Incubation of glucose-6-phosphate dehydrogenase with [3H]PLP-AMP followed by borohydride reduction shows that incorporation of 0.85 mol of PLP-AMP per mol of enzyme subunit is required for complete inactivation. Both glucose 6-phosphate and NAD+ protect against this covalent modification. The proteolysis of the modified enzyme and isolation and sequencing of the labeled peptides revealed that Lys-21 and Lys-343 are the sites of PLP-AMP interaction and that glucose 6-phosphate and NAD+ protect both lysyl residues against modification. Pyridoxal 5'-phosphate (PLP) also modifies Lys-21 and probably Lys-343. Lys-21 is part of a highly conserved region that is present in all glucose-6-phosphate dehydrogenases that have been sequenced. Lys-343 corresponds to an arginyl residue in other glucose-6-phosphate dehydrogenases and is in a region that is less homologous with those enzymes. PLP-AMP and PLP are believed to interact with L. mesenteroides glucose-6-phosphate dehydrogenase at the glucose 6-phosphate binding site. Simultaneous binding of NAD+ induces conformational changes (Kurlandsky, S. B., Hilburger, A. C., and Levy, H. R. (1988) Arch. Biochem. Biophys. 264, 93-102) that are postulated to interfere with Schiff's-base formation with PLP or PLP-AMP. One or both of the lysyl residues covalently modified by PLP or PLP-AMP may be located in regions of the enzyme undergoing the NAD(+)-induced conformational changes.     

1L-myo-inositol 1-phosphate synthase from pollen of Lilium longiflorum. An ordered sequential mechanism

1L-Inositol 1-phosphate synthase (EC 5.5.1.4) devoid of bound NAD+ was isolated from mature pollen of Lilium longiflorum ( Easter lily ). The enzyme has a molecular weight of 157,000 +/- 15,000 and a subunit weight of 61,000 +/- 5,000. Kinetic studies of the uninhibited reaction and of inhibition by 2-deoxy-D-glucose 6-phosphate and NADH show the reaction to be ordered sequential with NAD+ adding first. The Michaelis constants for NAD+ and D-glucose 6-phosphate are 2.4 and 65 microM, respectively. The Ki for 2-deoxy-D-glucose 6-phosphate was 8.7 and 2.0 microM, respectively, when D-glucose 6-phosphate or NAD+ was varied. The Ki for NADH and variable NAD+ was 4.7 microM and, for NADH and variable D-glucose 6-phosphate, 3.9 microM.     

The specificity of induced conformational changes. The case of yeast glyceraldehyde-3-phosphate dehydrogenase.

The specificity of induced conformational changes and of the probes used to detect them has been investigated in yeast glyceraldehyde-3-phosphate dehydrogenase. Cyanylation of the active-site SH groups in two of the four identical subunits of glyceraldehyde-3-phosphate dehydrogenase has no effect on reactivity of the unmodified SH groups toward the cyanylating reagent (2-nitro-5-thiocyanogenzoic acid, NTCB) but results in total loss of catalytic activity. Cyanylation of the dicarboxamidomethylated enzyme was four orders of magnitude slower than with the unmodified enzyme in contrast to cyanylation of the dicyanylated enzyme. Cyanylation by NTCB as well as alkylation by iodoacetate and acylation with beta-(2-furyl)acryloyl phosphate are enhanced in the presence of NAD+ while alkylation by iodoacetamide is inhibited by NAD+. In the absence of NAD+, hydrolysis of the acylated enzyme is faster than phosphorolysis while the reverse is true in the presence of NAD+. NAD+ accelerates hydrolysis of the 3-phosphoglyceroylated enzyme about 60-fold but decreases the rate of hydrolysis of the furylacryloylated enzyme by a factor of 17. Other examples of the specificity of the induced conformational changes and the probes are described. The conformational changes induced by NAD+ make the protein specifically reactive toward its physiological substrates and less reactive toward extraneous competing compounds.     

Evidence for ligand-induced conformational changes in rabbit-muscle glyceraldehyde-3-phosphate dehydrogenase.

The tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle binds NAD+ and some of its analogues in a negatively cooperative manner, whereas other NAD+ analogues bind non-cooperatively to this enzyme. Subsequent to alkylation of a fraction of the active sites of the enzyme with the fluorescent SH reagent N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine, it was found that the alkylated sites bind NAD+ and NAD+ analogues with a markedly reduced affinity as compared with non-alkylated sites. It was therefore feasible to measure the fluorescence and the circular polarization of the luminescence of the enzyme-bound alkyl groups as a function of binding of NAD+ and of NAD+ analogues to the non-alkylated sites. The changes observed indicate that ligand binding to the non-alkylated sites induces changes in the fluorescence properties of the alkyl groups bound to neighbouring subunits, most likely through the protein moiety. The nature of these changes appears to depend on the structure of the coenzyme analogue. The binding of the non-cooperative binders acetyl-pyridine--adenine dinucleotide, ATP and ADP-ribose induce different conformational changes in the neighbouring vacant subunit, as monitored by the spectroscopic properties of the bound alkyl group. These results in conjunction with other data support the view that the negative cooperativity in NAD+ binding to glyceraldehyde-3-phosphate dehydrogenase results from ligand-induced conformational changes. Furthermore, these results further support the view that subtle structural changes in the coenzyme molecule determine the nature of the conformational changes induced within the enzyme tetramer.     

Factors affecting coenzyme binding and subunit interactions in glyceraldehyde-3-phosphate dehydrogenase.

There is no evidence, at pH 9.4, of negative cooperativity in the binding of NAD+ or NADH to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phorphorylating), EC 1.2.1.12) nor in the binding of acetyl pyridine adenine dinucleotide at pH 7.6 and ph 9.4. The binding of NAD+ to carboxymethylated enzyme at pH 7.6 and pH 9.4 also occurs without cooperativity. The possible implications of these findings for the involvement of ionising groups in the enzyme in the subunit interactions responsible for negative cooperativity, previously reported for coenzyme binding at pH 7.4--8.6, are discussed.     

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.     

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.     

Site-directed mutagenesis of rat liver S-adenosylhomocysteinase. Effect of conversion of aspartic acid 244 to glutamic acid on coenzyme binding

Aspartic acid 244 that occurs at the putative NAD(+)-binding site of rat liver S-adenosylhomocysteinase was replaced by glutamic acid by oligonucleotide-directed mutagenesis. The mutant enzyme was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel permeation chromatography showed that the purified mutant enzyme was a tetramer as is the wild-type enzyme. In contrast to the wild-type enzyme, which possesses 1 mol of tightly bound NAD+ per mol of enzyme subunit, the mutant enzyme had only 0.05 mol of NAD+ but contained about 0.6 mol each of NADH and adenine per mol of subunit. The mutant enzyme, after removal of the bound compounds by acid-ammonium sulfate treatment, exhibited S-adenosylhomocysteinase activity when assayed in the presence of NAD+. From the appearance of activity as a function of NAD+ concentration, the enzyme was shown to bind NAD+ with a Kd of 23.0 microM at 25 degrees C, a value greater than 280-fold greater than that of the wild-type enzyme. In the presence of a saturating concentration of NAD+, the mutant enzyme showed apparent Km values for substrates similar to those of the wild-type enzyme. Moderate decreases of 8- and 15-fold were observed in Vmax values for the synthetic and hydrolytic directions, respectively. These results indicate the importance of Asp-244 in binding NAD+, and are consistent with the idea that the region of S-adenosylhomocysteinase from residues 213 to 244 is part of the NAD+ binding site. This region has structural features characteristic of the dinucleotide-binding domains of NAD(+)- and FAD-binding proteins (Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P.S., Jr., Aksamit, R.R., Unson, C.G., and Cantoni, G.L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 719-723).     

Purification and properties of sorbitol dehydrogenase from mouse liver

1. The sorbitol dehydrogenase (L-iditol: NAD oxidoreductase, EC 1.1.1.14) from mouse liver has been purified to homogeneity. 2. The enzyme has a mol. wt of 140,000 and is composed of four identical subunits of mol. wt 35,000. 3. the purified enzyme catalyses both sorbitol oxidation and fructose reduction. 4. It is specific for NAD+ (NADH) and does not function with NADP+ (NADPH). 5. The Michaelis constants for sorbitol, fructose, NAD+ and NADPH are 1.54 and 154 mM, 58.8 and 15 microM, respectively. 6. The enzyme is SH-group reagent sensitive and is strongly inhibited by 1,10-phenanthroline.     

Inactivation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by koningic acid

Koningic acid, a sesquiterpene antibiotic, is a specific inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). In the presence of 3 mM of NAD+, koningic acid irreversibly inactivated the enzyme in a time-dependent manner. The pseudo-first-order rate constant for inactivation (kapp) was dependent on koningic acid concentration in saturate manner, indicating koningic acid and enzyme formed a reversible complex prior to the formation of an inactive, irreversible complex; the inactivation rate (k 3) was 5.5.10(-2) s-1, with a dissociation constant for inactivation (Kinact) of 1.6 microM. The inhibition was competitive against glyceraldehyde 3-phosphate with a Ki of 1.1 microM, where the Km for glyceraldehyde 3-phosphate was 90 microM. Koningic acid inhibition was uncompetitive with respect to NAD+. The presence of NAD+ accelerated the inactivation. In its absence, the charcoal-treated NAD+-free enzyme showed a 220-fold decrease in apparent rate constant for inactivation, indicating that koningic acid sequentially binds to the enzyme next to NAD+. The enzyme, a tetramer, was inactivated when maximum two sulfhydryl groups, possibly cysteine residues at the active sites of the enzyme, were modified by the binding of koningic acid. These observations demonstrate that koningic acid is an active-site-directed inhibitor which reacts predominantly with the NAD+-enzyme complex.     

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.     

Characterization of two D-glyceraldehyde-3-phosphate dehydrogenases from the extremely thermophilic archaebacterium Thermoproteus tenax

Thermoproteus tenax possesses two different glyceraldehyde-3-phosphate dehydrogenases, one specific for NADP+ and the other for NAD+. NADP(H) inhibits the NAD+-specific enzyme competetively with respect to NAD+ whereas NAD(H) virtually does not interact with the NADP+-specific enzyme. Both enzymes represent homomeric tetramers with subunit molecular masses of 39 kDa (NADP+-specific enzyme) and 49 kDa (NAD+-specific enzyme), respectively. The NADP+-specific enzyme shows significant homology to the known glyceraldehyde-3-phosphate dehydrogenases from eubacteria and eukaryotes as indicated by partial sequencing. The enzymes are thermostable, the NADP+-specific enzyme with a half-life of 35 min at 100 degrees C, the NAD+-specific enzyme with a half-line of greater than or equal to 20 min at 100 degrees C, depending on the protein concentration. Both enzymes show conformational and functional changes at 60-70 degrees C.     

Evidence for an essential lysine in glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

1. Pyridoxal 5'-phosphate inhibits glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides reversibly which Ki equals 0.04-0.06 mM. 2. This inhibition is competitive with respect to glucose 6-phosphate and non-competitive with respect to NADP+ or NAD+. Interaction between enzyme and excess pyridoxal 5'-phosphate follows pseudo-first-order kinetics and indicates that one molecule of inhibitor reacts with each active unit of enzyme. 3. Substrate and coenzyme protect the enzyme from inhibition by pyridoxal 5'-phosphate. Dissociation constants for NADP+ and glucose 6-phosphate were determined from their effects on the kinetics of enzyme--inhibitor interaction. 4. Reaction of the enzyme with pyridoxal 5'-phosphate produces a typical Schiff-base absorbance peak at 430 nm. Subsequent reduction with sodium borohydride leads to spectral changes characteristic for the formation of a secondary amine. 5. The irreversibly inactivated enzyme thus produced contains two moles of inhibitor per mole of enzyme (two subunits per mole). After protein hydrolysis, N-6-pyridoxyllysine can be identified by paper chromatography. 6. The enzyme is inhibited irreversibly by 1-fluoro-2,4-dinitrobenzene, even in the presence of excess 2-mercaptoethanol. At least one dinitrophenyl group is bound per active unit of enzyme; 4 to 5 moles of dinitrophenyl group are bound per mole of enzyme. NADP+ AND GLUCOSE 6-PHOSPHATE PROTECT AGAINST INHIBITION BY 1-FLUORO-2,4-DINITROBENZENE. The absorption spectrum of dinitrophenyl-enzyme corresponds to that for dinitrophenylated amino groups. 7. These studies indicate that there is an essential lysine at the active site of the enzyme. It is suggested that the function of this lysine is to bind glucose 6-phosphate. 8. It is proposed that a group of "active lysine" proteins may exist (in analogy with the "active serine" enzymes), which share a common structural feature at their substrate-binding site and to which pyridoxal 5'-phosphate binds specifically.     

Dehydroquinate synthase in Bacillus subtilis. An enzyme associated with chorismate synthase and flavin reductase.

Dehydroquinate synthase, the enzyme which catalyzes the conversion of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) to 5-dehydroquinate, has been purified from Bacillus subtilis in association with chorismate synthase and NADPH-dependent flavin reductase. The enzyme was only active when associated with chorismate synthase, whereas the flavin reductase could be separated from the complex with retention of dehydroquinate synthase activity. The enzyme requires NAD and either Co2+ or Mn2+ for maximal activity. The activity was completely inhibited by EDTA. The Km of the enzyme for DAHP, NAD, and Co2+ were estimated to be 1.3 X 10(-4), 5.5 X 10(-5), and 5.5 X 10(-5) M, respectively. Enzyme activity was completely inhibited by NADH and the inhibition was not reversed by the addition of NAD, NADPH and NADP were not inhibitory. The enzyme was unstable to heat and lost all activity at 55 degrees C. A protein fraction which did not adsorb to phosphocellulose was found to inhibit the enzyme.     

Chirality of the hydrogen transfer to the coenzyme catalyzed by ribitol dehydrogenase from Klebsiella pneumoniae and D-mannitol 1-phosphate dehydrogenase from Escherichia coli.

The stereochemistry of the hydrogen transfer to NAD catalyzed by ribitol dehydrogenase (ribitol:NAD 2-oxidoreductase, EC 1.1.1.56) from Klebsiella pneumoniae and D-mannitol-1-phosphate dehydrogenase (D-mannitol-1-phosphate:NAD 2-oxidoreductase, EC 1.1.1.17) from Escherichia coli was investigated. [4-3H]NAD was enzymatically reduced with nonlabelled ribitol in the presence of ribitol dehydrogenase and with nonlabelled D-mannitol 1-phosphate and D-mannitol 1-phosphate dehydrogenase, respectively. In both cases the [4-3H]-NADH produced was isolated and the chirality at the C-4 position determined. It was found that after the transfer of hydride, the label was in both reactions exclusively confined to the (4R) position of the newly formed [4-3H]NADH. In order to explain these results, the hydrogen transferred from the nonlabelled substrates to [4-3H]NAD must have entered the (4S) position of the nicotinamide ring. These data indicate for both investigated inducible dehydrogenases a classification as B or (S) type enzymes. Ribitol also can be dehydrogenated by the constitutive A-type L-iditol dehydrogenase (L-iditol:NAD 5-oxidoreductase, EC 1.1.1.14) from sheep liver. When L-iditol dehydrogenase utilizes ribitol as hydrogen donor, the same A-type classification for this oxidoreductase, as expected, holds true. For the first time, opposite chirality of hydrogen transfer to NAD in one organic reaction--ribitol + NAD = D-ribu + NADH + H--is observed when two different dehydrogenases, the inducible ribitol dehydrogenase from K. pneumoniae and the constitutive L-iditol dehydrogenase from sheep liver, are used as enzymes. This result contradicts the previous generalization that the chirality of hydrogen transfer to the coenzyme for the same reaction is independent of the source of the catalyzing enzyme.     

Mannitol-1-phosphate dehydrogenase of Escherichia coli. Chemical properties and binding of substrates.

Mannitol-1-phosphate dehydrogenase was purified to homogeneity, and some chemical and physical properties were examined. The isoelectric point is 4.19. Amino acid analysis and polyacrylamide-gel electrophoresis in presence of SDS indicate a subunit Mr of about 22,000, whereas gel filtration and electrophoresis of the native enzyme indicate an Mr of 45,000. Thus the enzyme is a dimer. Amino acid analysis showed cysteine, tyrosine, histidine and tryptophan to be present in low quantities, one, three, four and four residues per subunit respectively. The zinc content is not significant to activity. The enzyme is inactivated (greater than 99%) by reaction of 5,5'-dithiobis-(2-nitrobenzoate) with the single thiol group; the inactivation rate depends hyperbolically on reagent concentration, indicating non-covalent binding of the reagent before covalent modification. The pH-dependence indicated a pKa greater than 10.5 for the thiol group. Coenzymes (NAD+ and NADH) at saturating concentrations protect completely against reaction with 5,5'-dithiobis-(2-nitrobenzoate), and substrates (mannitol 1-phosphate, fructose 6-phosphate) protect strongly but not completely. These results suggest that the thiol group is near the catalytic site, and indicate that substrates as well as coenzymes bind to free enzyme. Dissociation constants were determined from these protective effects: 0.6 +/- 0.1 microM for NADH, 0.2 +/- 0.03 mM for NAD+, 9 +/- 3 microM for mannitol 1-phosphate, 0.06 +/- 0.03 mM for fructose 6-phosphate. The binding order for reaction thus may be random for mannitol 1-phosphate oxidation, though ordered for fructose 6-phosphate reduction. Coenzyme and substrate binding in the E X NADH-mannitol 1-phosphate complex is weaker than in the binary complexes, though in the E X NADH+-fructose 6-phosphate complex binding is stronger.     

Biosynthesis of vitamin B6 in Rhizobium: in vitro synthesis of pyridoxine from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine

Pyridoxine (vitamin B6) in Rhizobium is synthesized from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine. To define the pathway enzymatically, we established an enzyme reaction system with a crude enzyme solution of R. meliloti IFO14782. The enzyme reaction system required NAD+, NADP+, and ATP as coenzymes, and differed from the E. coli enzyme reaction system comprising PdxA and PdxJ proteins, which requires only NAD+ for formation of pyridoxine 5'-phosphate from 1-deoxy-D-xylulose 5-phosphate and 4-(phosphohydroxy)-L-threonine.     

Studies of coenzyme binding to rabbit muscle glyceraldehyde-3-phoshate dehydrogenase.

The binding of NAD+, NADH and adenosine diphosphoribose (Ado-PP-Rib) to a stable, highly active and nucleotide-free preparation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) has been studied. All three nucleotides quench the protein fluorescence to the same extent when they bind to the enzyme, and this property has been used to measure the dissociation constants for the two high-affinity binding sites for the nucleotides. The results indicate negative interactions between, or non-identify of, these two binding sites, to which NAD+ and NADH bind with similar affinity. The binding of NAD+ to the enzyme has been studied by spectrophotometric titrations at 360 nm. It appears that the binding of NAD+ to each of the four subunits of the enzyme contributes equally to the intensity of this 'Racker' band. The dissociation constants associated with the binding of the third and fourth molecules of NAD+ estimated from such titrations confirm some previous estimates. The binding of NADH to the enzyme causes a decrease of intensity of the absorbance of the coenzyme at 340 nm, and the dissociation constants for binding of the third and fourth molecules of NADH have been estimated from spectrophotometric titrations. They are the same as those for NAD+. Judging by the apparent dissociation constants, negative interactions on binding the third molecule of NAD+ or NADH are more marked than those associated with the binding of the second and fourth molecules, suggesting that a major conformational change occurs at half-saturation of the tetramer with coenzyme.     

Sturgeon glyceraldehyde-3-phosphate dehydrogenase.

The formation of binary complexes between sturgeon apoglyceralddhyde-3-phosphate dehydrogenase, coenzymes (NAD+ and NADH) and substrates (phosphate, glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate) has been studied spectrophotometrically and spectrofluorometrica-ly. Coenzyme binding to the apoenzyme can be characterized by several distinct spectroscopic properties: (a) the low intensity absorption band centered at 360 nm which is specific of NAD+ binding (Racker band); (b) the quenching of the enzyme fluorescence upon coenzyme binding; (c) the quenching of the fluorescence of the dihydronicotinamide moiety of the reduced coenzyme (NADH); (D) the hypochromicity and the red shift of the absorption band of NADH centered at 338 nm; (e) the coenzyme-induced difference spectra in the enzyme absorbance region. The analysis of these spectroscopic properties shows that up to four molecules of coenzyme are bound per molecule of enzyme tetramer. In every case, each successively bound coenzyme molecule contributes identically to the total observed change. Two classes of binding sites are apparent at lower temperatures for NAD+ Binding. Similarly, the binding of NADH seems to involve two distinct classes of binding sites. The excitation fluorescence spectra of NADH in the binary complex shows a component centered at 260 nm as in aqueous solution. This is consistent with a "folded" conformation of the reduced coenzyme in the binary complex, contradictory to crystallographic results. Possible reasons for this discrepancy are discussed. Binding of phosphorylated substrates and orthophosphate induce similar difference spectra in the enzyme absorbance region. No anticooperativity is detectable in the binding of glyceraldehyde 3-phosphate. These results are discussed in light of recent crystallographic studies on glyceraldehyde-3-phosphate dehydrogenases.