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.     

Preparation and properties of rabbit-muscle glyceraldehyde-phosphate dehydrogenase with equal binding parameters for the third and fourth NAD+ molecules.

1. A method of preparing rabbit-muscle glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) is described which yields a preparation differing in important respects from those previously described and resembling the enzyme isolated from sturgeon muscle. 2. Direct binding measurements at 25 degrees C by equilibrium gel filtration fit dissociation constants for the first two molecules that are too low to be measured by this technique and 0.9 micrometer for the third and fourth molecules. The dissociation constant of the fourth molecule is much lower than that previously reported for the rabbit-muscle enzyme. 3. In contrast to previous results with the rabbit-muscle enzyme, the increase in absorbance at 360 nm between three and four molecules of NAD+ bound to the enzyme was, within experimental error, the same as that with each of the first three molecules. 4. Data on the quenching of the protein fluorescence by NAD+ at 15 degrees C at different enzyme concentrations closely fit dissociation constants of 0.028 micrometer for the first two molecules and 0.27 micrometer for the third and fourth molecules.     

NAD+ and NAD+ analogues in horse liver alcohol dehydrogenase. Relationship between reactivity and conformation simulated with molecular mechanics

In the present study we show that the enzymatic activity of the coenzyme nicotinamide adenine dinucleotide (NAD+) and its analogues (C(O)NH2 replaced by C(S)NH2, C(O)CH3, C(O)H and CN) with horse liver alcohol dehydrogenase (LADH) (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) can be rationalized by their conformation in the active site determined with molecular mechanics (AMBER, assisted model building with energy refinement). In order to establish the relation between the hydride transfer rate and the conformation of the NAD+ and its analogues, kinetic experiments with the poor substrate isopropanol were carried out. It appears that the enzymatic activity can be readily explained by the geometry of the pyridinium ring, in particular the magnitude of the 'out-of-plane' rotation of the carboxamide side chain (or analogues). The latter is nicely illustrated in the case of 3-cyanopyridine adenine dinucleotide which lacks any 'out-of-plane' rotation and concomitantly exhibits no significant enzymatic activity.     

Hysteresis and conformational drift of pressure-dissociated glyceraldehydephosphate dehydrogenase

Pressure dissociation of yeast glyceraldehydephosphate dehydrogenase (GAPDH) was studied by fluorescence spectroscopy. Observations in the range of -5 to 30 degrees C indicate that monomer association into the tetramer proceeds with an enthalpy change of -14 kcal mol-1 and a large increase in entropy which at 25 degrees C amounts to 18 kcal mol-1. The large conformational drift and the low-temperature stability of the tetramer recovered after decompression facilitated a comparison of its properties with those of the native tetramer. Significant differences in absorption and fluorescence-excitation polarization spectra, yield of tryptophan fluorescence, and binding of anilinonaphthalenesulfonate and NADH were observed. At 0 degree C the standard free energies of association of the monomers into the native and drifted tetramers were respectively -32 and -29 kcal mol-1. The volume change upon association measured from the pressure span of the compression curves was 200-230 mL mol-1 but four times as large when derived from the displacement of the compression curves with total protein concentration. This large discrepancy can be explained by the existence in the native tetramer population of a distribution of free energies of association with a dispersion from the mean of about 6 kcal mol-1. At 0 degree C and 1 bar ATP and ADP decreased the stability of the GAPDH tetramer by changes in free energy of association of +3.7 and +4.1 kcal mol-1, respectively. NAD and c-AMP stabilized it by -2.3 and -1.3 kcal mol-1. The variation in sign and magnitude of the ligand-induced changes in free energy of association observed in this case, and previously in hexokinase [Ruan, K., & Weber, G. (1988) Biochemistry 27, 3295], and the heterogeneity of the free energy of association of GAPDH, revealed as indicated above, lead to the conclusion that oligomeric aggregates exist in a variety of conformations that depend upon the protein concentration, temperature, pressure, and the presence of specific ligands. The multiplicity of species revealed by the energetics raises questions about the significance of the structures of oligomeric proteins determined by X-ray crystallography.     

The mechanism of adduct formation between NAD+ and pyruvate bound to pig heart lactate dehydrogenase.

1. The rate of adduct formation between NAD+ and enol-pyruvate at the active site of lactate dehydrogenase is determined by the rate of enolization of pyruvate in solution. 2. The proportion of enol-pyruvate solutions is less than 0.01%. 3. The overall dissociation constant of adduct formation is less than 5 X 10(-8) M for pig heart lactate dehydrogenase at pH 7.0. 4. The unusual kinetics for adduct formation previously observed in the case of rabbit muscle lactate dehydrogenase [Griffin & Criddle (1970) Biochemistry 9, 1195--1205] may be attributed to the concentration of enol-pyruvate in solution being considerably less than the concentration of enzyme.     

NAD+-dependent retinol dehydrogenase in liver microsomes

A microsomal NAD+-dependent retinol dehydrogenase is being described with optimal activity at physiological pH. The enzyme was present in liver microsomes of rats and also in a strain of deermice which lacks the cytosolic retinol dehydrogenase. Unlike the latter enzyme, the microsomal retinol dehydrogenase was not inhibited by either ethanol or 4-methylpyrazole; its activity was insensitive to CO and not oxygen dependent, in contradistinction with that of the microsomal cytochrome P-450 and NADPH-dependent retinol oxidase. Chronic ethanol consumption resulted in an increased activity of the microsomal retinol dehydrogenase which may contribute to hepatic retinol depletion, especially in view of the insensitivity of the enzyme to ethanol inhibition.     

NAD+ analogue binding to glyceraldehyde-3-phosphate dehydrogenase

A series of NAD+ analogues, modified on the pyridinium ring, have been tested for their enzymic properties in reactions with D-glyceraldehyde-3-phosphate dehydrogenase form sturgeon muscle, rabbit muscle and Bacillus stearothermophilus. The observed activity, inhibition and binding data are correlated to the structure of the enzyme and coenzyme analogue by model building on a Vector General interactive graphic display system using coordinates from the B. stearothermophilus holoenzyme structure. Most of the analogues with substituents in the pyridinium-3 position could be bound to glyceraldehyde-3-phosphate dehydrogenase, either in manner similar to NAD+ or in a completely different way with the substituted pyridinium ring rotated 110 degrees or more around the glycosidic bond. This indicates different possible modes of binding of NAD+ analogues within the pyridinium binding subsite. Analogues with substituents in the pyridinium-4 position are shown to be weakly bound to glyceraldehyde-3-phosphate dehydrogenase. This is explained by a strong interaction of the substituent in the 4 position with the residues Asn-313 and Cys-149.     

Subunit interactions of yeast NAD+-specific isocitrate dehydrogenase

Yeast mitochondrial NAD(+)-specific isocitrate dehydrogenase is an octamer composed of four each of two nonidentical but related subunits designated IDH1 and IDH2. IDH2 was previously shown to contain the catalytic site, whereas IDH1 contributes regulatory properties including cooperativity with respect to isocitrate and allosteric activation by AMP. In this study, interactions between IDH1 and IDH2 were detected using the yeast two-hybrid system, but interactions between identical subunit polypeptides were not detected with this or other methods. A model for heterodimeric interactions between the subunits is therefore proposed for this enzyme. A corollary of this model, based on the three-dimensional structure of the homologous enzyme from Escherichia coli, is that some interactions between subunits occur at isocitrate binding sites. Based on this model, two residues (Lys-183 and Asp-217) in the regulatory IDH1 subunit were predicted to be important in the catalytic site of IDH2. We found that individually replacing these residues with alanine results in mutant enzymes that exhibit a drastic reduction in catalysis both in vitro and in vivo. Also based on this model, the two analogous residues (Lys-189 and Asp-222) of the catalytic IDH2 subunit were predicted to contribute to the regulatory site of IDH1. A K189A substitution in IDH2 was found to produce a decrease in activation of the enzyme by AMP and a loss of cooperativity with respect to isocitrate. A D222A substitution in IDH2 produces similar regulatory defects and a substantial reduction in V(max) in the absence of AMP. Collectively, these results suggest that the basic structural/functional unit of yeast isocitrate dehydrogenase is a heterodimer of IDH1 and IDH2 subunits and that each subunit contributes to the isocitrate binding site of the other.     

Kinetic mechanism of Halobacterium halobium NAD+-glutamate dehydrogenase

The kinetic mechanism of Halobacterium halobium NAD+-glutamate dehydrogenase (EC 1.4.1.3) has been investigated at pH 9.0, 3 M NaCl and 40 degrees C in both directions, by initial rate and inhibition studies. The results of the initial rate studies indicate that the mechanism is sequential with respect to substrate addition. The inhibition patterns obtained with halophilic NAD+-glutamate dehydrogenase are not consistent with a simple ordered mechanism without modification. They can, however, be reconciled with this type of mechanism by postulating an appropriate abortive complex.