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.     

Studies on coenzyme binding to glyceraldehyde-3-phosphate dehydrogenase.

Tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus has been crystallized as hexagonal plates, $\text{P3}{}_1\text{21, a = b = 64.6}\text{A}\text{?}\text{, c = 238.8}\text{A}\text{?}$, with the dimeric molecule (molecular weight, 90,000) occupying two crystallographic asymmetric units (Reid et al., 1973). Three heavy-atom derivatives have been identified and X-ray diffraction measurements have been made to 2.7 Å resolution, using the oscillation method. The three heavy-atom derivatives were methyl mercury (two sites, half occupied, 3 Å apart), uranyl acetate (single fully occupied site) and chloroplatinite PtCl₄^2? (three sites of differing occupancy). The results were used to compute an electron density map at 2.7 Å resolution, which shows the monomer as a unit of about 60 Å × 60 Å × 40 Å. The maximum dimension of the dimer is about 130 Å. Most of the polypeptide chain has been traced uniquely. It includes five α-helices more than 12 Å long and several shorter helices. A six-stranded pleated-sheet structure lies in the centre of each subunit. The catalytic site of the enzyme is believed to be adjacent to the mercury-binding group.     

Mechanism of glyceraldehyde-3-phosphate transfer from aldolase to glyceraldehyde-3-phosphate dehydrogenase

The catalytic interaction of glyceraldehyde-3-phosphate dehydrogenase with glyceraldehyde 3-phosphate has been examined by transient-state kinetic methods. The results confirm previous reports that the apparent Km for oxidative phosphorylation of glyceraldehyde 3-phosphate decreases at least 50-fold when the substrate is generated in a coupled reaction system through the action of aldolase on fructose 1,6-bisphosphate, but lend no support to the proposal that glyceraldehyde 3-phosphate is directly transferred between the two enzymes without prior release to the reaction medium. A theoretical analysis is presented which shows that the kinetic behaviour of the coupled two-enzyme system is compatible in all respects tested with a free-diffusion mechanism for the transfer of glyceraldehyde 3-phosphate from the producing enzyme to the consuming one.     

Oxidation of glyceraldehyde-3-phosphate dehydrogenase enhances its binding to nucleic acids

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a protein with various activities far from its enzymatic function. Here, we showed that the oxidation of SH-groups of the active site of GAPDH enhanced its binding with total transfer RNA or with total DNA. Both NAD and NADH-the cofactors of GAPDH-inhibited the GAPDH-RNA (DNA) interaction, though NAD was much less effective than NADH in the case of oxidized GAPDH. Oxidation of GAPDH strongly decreased its affinity to NAD but not to NADH. Immobilized tetramers of GAPDH dissociated into dimers during the incubation with total RNA but not DNA. The staining of HeLa cells with monoclonal antibodies specific to dimers, monomers or the denatured form of GAPDH revealed the condensation of non-native forms of GAPDH in the nucleus. The role of oxidation of GAPDH in the regulation of the quaternary structure of the enzyme and in its interaction with nucleic acids is discussed.     

Spin label and lanthanide binding sites on glyceraldehyde-3-phosphate dehydrogenase.

The electron spin resonance spectrum of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase spin-labelled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl has two components. One component is due to a spin label highly immobilized on the enzyme surface and the other to a nitroxyl group able to tumble more rapidly. The spin-labelled enzyme is inactive. Selective modification of the active site cysteine residue (149) and determinations of total sulphydryl content implicate this residue as the site of the immobile spin-label. The mobile spin label is attached to another sulphydryl group. Crystallographic studies on the human muscle enzyme (Watson, H.C., Duee, E. and Mercer, W.D. (1972) Nat. New Biol., 240, 130) have located a binding site for samarium ion in the active centre. Addition of the paramagnetic gadolinium ion to spin-labelled enzyme reduces the intensity of both the spin label signals (by 72% for the mobile and by 11% for the immobile component). This indicates that the metal ion site (Kd equals 0.7 mM) is close to both types of spin label. Measurements of the effect of gadolinium-protein binding on the relaxation rate of solvent water protons enable the enzyme-bound spin label-metal ion distances to be tentatively estimated as 15 angstrom.     

Ascorbate-induced oxidation of glyceraldehyde-3-phosphate dehydrogenase

Oxidation of the essential cysteins of glyceraldehyde-3-phosphate dehydrogenase into the sulfenic acid derivatives was observed in the presence of ascorbate, resulting in a decrease in the dehydrogenase activity and the appearance of the acylphosphatase activity. The oxidation was promoted by EDTA, NAD(+), and phosphate, and blocked in the presence of deferoxamine. The ascorbate-induced oxidation was suppressed in the presence of catalase, suggesting the accumulation of hydrogen peroxide in the conditions employed. The data indicate the metal-mediated mechanism of the oxidation due to the presence of metal traces in the reaction medium. Physiological importance of the mildly oxidized GAPDH is discussed in terms of its ability to uncouple glycolysis and to decrease the ATP level in the cell.     

Temperature studies of glyceraldehyde-3-phosphate dehydrogenase binding to liposomes using fluorescence technique.

Interaction of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase with negatively charged liposomes was investigated as a function of temperature. This interaction affects the temperature-dependent conformational transition in the enzyme and exerts stabilizing effect on the protein structure. It can be seen from the fluorescence quenching experiments that the accessibility of tryptophanyl residues and isoindol probe fluorophores (covalently bound with the protein amino groups) for a dynamic quencher, acrylamide, is altered upon binding. This accessibility represented by effective quenching constant (Keff) strongly depends on temperature for unmodified enzyme and for the enzyme adsorbed on liposomes, it is nearly constant over a wide range of temperatures.     

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.     

Sequestration of glyceraldehyde-3-phosphate dehydrogenase to aggregates formed by mutant huntingtin

Glyceraldehyde-3-phosphate dehydrogenase （GAPDH） has been reported to interact with proteins containing the polyglutamine （polyQ） domain. The present study was undertaken to evaluate the potential contributions of the polyQ and polyproline （polyP） domains to the co-localization of mutant huntingtin （htt） and GAPDH. Overexpression of N-terminal htt （1-969 amino acids） with 100Q and 46Q （httl-969- 100Q and httl-969-46Q, mutant htt） in human mammary gland carcinoma MCF-7 cells formed more htt aggregates than that of httl-969-18Q （wild-type htt）. The co-localization of GAPDH with htt aggregates was found in the cells expressing mutant but not wild-type htt. Deletion of the polyP region in the N-terminal htt had no effect on the co-localization of GAPDH and mutant htt aggregates. These results suggest that the polyQ domain, but not the polyP domain, plays a role in the sequestration of GAPDH to aggregates by mutant htt. This effect might contribute to the dysfunction of neurons caused by mutant htt in Huntington＇s disease.     

Lack of binding of glyceraldehyde-3-phosphate dehydrogenase to erythrocyte membranes under in vivo conditions

A filtration method is described for separating membrane-free cytoplasm from concentrated erythrocyte haemolysates. The method has been used to assess glyceraldehyde-3-phosphate dehydrogenase binding to erythrocyte membranes. The relative amounts of glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase in the cytoplasm (either oxygenated or deoxygenated) indicate there is no detectable binding of glyceraldehyde-3-phosphate dehydrogenase to the membranes under physiological conditions.