Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It is shown that the modulation in the negative cooperativity of ligand binding by another, competing ligand that binds noncooperatively is accounted for exclusively by the ligand-induced sequential model. It is therefore suggested that whenever such a phenomenon is observed it argues strongly in favor of the sequential model. The advantages and limitations of this approach are evaluated. The binding of the coenzymes NAD+ and nicotinamide-1-N6-ethenoadenine dinucleotide to rabbit muscle apo-glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating; EC 1.2.1.12] exhibits strong negative cooperativity, whereas acetylpyridine adenine dinucleotide, ATP, and ADP-ribose bind noncooperatively to the NAD+ sites. The strong abolished in the presence of acetylpyridine adenine dinucleotide and strongly weakened by ATP, ADP, and AMP, but was not affected by addition of ADP-ribose. These findings demonstrate that the negative cooperativity in coenzyme binding to this enzyme results from sequential conformational changes and exclude the pre-existent asymmetry model as a possible explanation. These results also support the view that the structure of the pyridine moiety of the coenzyme analogs plays a role in orienting the adenine moiety at the adenine subsite, therefore affecting the cooperativity in the binding of the coenzyme analog which is mediated through the adenine subsites.
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PMID:Mechanism of negative cooperativity in glyceraldehyde-3-phosphate dehydrogenase deduced from ligand competition experiments. 693 45

1. The hybridization of rabbit muscle and yeast glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) was used to study the involvement of subunit interactions in NAD+ and NADH binding by these enzymes. 2. In the presence of 1 mM NAD+ or NADH no hybrid formation was observed with our preparations of the two enzymes. 3. The inhibition by NADH of the hybrid formation is shown to be a consequence of an unfavourable equilibrium of the hybridization process in the presence of NADH. 4. The inhibition by NAD+ of the hybrid formation is shown to be a consequence of both a shift in the equilibrium, as in the case of NADH, and a decrease in the rate of the dissociation of the enzymes. 5. The dimer of the yeast enzyme binds NAD+ or NADH with equal affinity irrespective of whether it is combined with another yeast dimer in the yeast tetramer or with a rabbit muscle dimer in the hybrid. 6. The binding of NAD+ and NADH to the dimer of the rabbit muscle enzyme is stronger in the rabbit muscle tetramer than in the hybrid; this explains the shift in the equilibrium of the hybridization process caused by these nucleotides. 7. Alkylation of the rabbit muscle enzyme with iodoacetate does not influence the hydridization process in the absence of nucleotides. 8. After alkylation of the rabbit muscle enzyme NADH cannot cause a large shift in the equilibrium of the hybridization process. 9. In accordance with this it was found that the binding of NADH (and NAD+) to the rabbit muscle enzyme is weakened by alkylation, whereas the binding of NADH to the alkylated rabbit muscle subunits is not affected strongly by the hydridization. 10. An attempt is made to combine the effects of nucleotides on the hybridization properties of the yeast enzyme and the alkylated or unalkylated rabbit muscle enzymes with the binding properties of all tetrameric species involved in the hybridization processes in a thermodynamic description of nucleotide binding and subunit interactions.
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PMID:Subunit interactions in glyceraldehyde-3-phosphate dehydrogenases. Their involvement in nucleotide binding and cooperativity. 700 89

Under conditions which cause dissociation of soluble tetrameric glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) into inactive dimers, immobilized apoenzymes from yeast and rat skeletal muscle coupled to CnBr-activated Sepharose via one subunit retain 50% of matrix-bound protein with unaltered specific activity. The solubilized dissociated species are inactive. Two molecules of NAD+ (NADH) firmly bound to the immobilized rat muscle tetramer can prevent the dissociation. Immobilized dimer was demonstrated to bind one molecule of coenzyme with high affinity. Using various combinations of immobilized and soluble rat muscle and yeast dimers, we succeeded in reconstituting tetramers, containing one molecule of NAD+ bound either to a matrix-linked or to a non-covalently bound dimer. In the latter case, the dissociation of the tetramer was completely prevented. This suggests that the binding of a single coenzyme molecule is sufficient to stabilize the interdimeric contacts provided the neighbouring dimer is stabilized independently. Such stabilization is produced by the covalent binding of one of the subunits comprising the dimer to the matrix. The structure of the dimer as a whole becomes resistant to the action of the dissociating agent. The effect appears to be cooperative and similar to that of NAD+ or NADH. The dissociation of the immobilized tetramer is, most likely, the result of conformational changes, affecting the structure of the non-covalently bound dimer. Any factor, capable of preventing these changes, would stabilize the interdimeric contacts. The latter conclusion is substantiated by the effect of specific antibodies, which prevent the dissociation of the immobilized tetramer by forming a complex with the dimer, non-covalently bound to the matrix. The evidence obtained in the present investigation supports the conclusion that the isolated dimer of glyceraldehyde-3-phosphate dehydrogenase represents a relatively independent structural and functional 'unit' of the enzyme. It can be stabilized in a catalytically active form by interactions other than those involved in inter-dimeric contacts in the tetramer. The kinetics of the association of immobilized and soluble dimers have been studied. Association rate constants were determined for homologous (yeast-yeast, rat-rat) and heterologous (yeast-rat, yeast-rabbit) dimer combinations. The binding of one molecule of specific antibody to the immobilized dimer was shown to increase the rate constant of association.
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PMID:Study of subunit interactions in immobilized D-glyceraldehyde-3-phosphate dehydrogenase. 700 90

Yeast glyceraldehyde-3-phosphate dehydrogenase was labeled in a photodependent reaction by the arylazido-beta-alanyl derivative of NAD+. This analogue was bound covalently to the enzyme and could be reduced in situ by the substrate glyceraldehyde-3-phosphate. That this derivative was bound to the active site in the proper orientation was shown by fluorescence experiments, from the retention of the enzymatic activity when the photolysis of the enzyme-analogue binary complex was carried out in the presence of NAD+. In the dark a non-photodependent competitive inhibition corresponding to a KI-value of 150 microM was observed. Thiol groups of the enzyme were not modified in the photolabeling reaction. Of the various arylazido-beta-alanyl nucleotide derivatives studied, the NADP+ derivative influenced the enzymatic activity to the greatest extent; this is probably due to an ionic bond between enzyme and nucleotide, in addition to the covalent bond of the photolytic reaction.
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PMID:Photodependent incorporation of arylazido-beta-alanyl-NAD+ into the coenzyme binding site of yeast glyceraldehyde-3-phosphate dehydrogenase. 702 21

The evidence for equivalent catalytic sites in tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle has been re-examined and found to be insufficient to exclude alternating or reciprocating sites models. Using a column centrifugation technique, lower limits have been set on the rates of binding and release of coenzyme, and on the ratio of the affinities of NAD+ and NADH. The binding to acyl enzyme has also been examined. The tightly bound NAD+ has been found to be reduced preferentially and kinetically competently when glyceraldehyde 3-phosphate is added, demonstrating the nonequivalence of the sites in the transient reduction of NAD+. The rate of release of the NADH formed rapidly from tightly bound NAD+ was monitored directly by using lactate dehydrogenase and pyruvate to regenerate NAD+. This rate was sufficiently rapid for the NADH formed from tightly bound NAD+ to be a catalytic intermediate. Although these and other results are consistent with a simple alternating sites model, additional approaches appear necessary to find if subunit catalytic cooperativity occurs with this enzyme.
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PMID:Subunit interaction in catalysis. Some experimental and theoretical approaches with glyceraldehyde-3-phosphate dehydrogenase. 708 42

A number of NAD+ analogs have been tested in their ability to form fluorescent derivatives when UV irradiated with the active site Cys-149 carboxymethylated GAPDH and this has been compared with their properties of acting as hydrogen acceptors and forming the Racker band. Among the analogs tested, NHD+, NGD+, APAD+ and epsilon NAD+ give positive results in all the above-mentioned reactions whereas alpha NAD+, NMN+ and CPAD+ are all negative. FPAD+ forms a fluorescent derivative on UV irradiation with the carboxymethylated enzyme but is inactive as a hydrogen acceptor and does not form the Racker band. This is probably due to thiohemiacetal formation of the pyridine 3-aldehyde of this derivative with the active site SH group required for both the latter 2 reactions. TPAD+, although active active as a hydrogen acceptor, does not form either a fluorescent derivative or a Racker band. The fact that for the great majority of the analogs, the property of forming fluorescent derivatives is in parallel with their hydrogen acceptor activity seems to show that the formation of the fluorescent derivative is indeed at the active site, and hence can be used as an intrinsic probe for the study of the conformation of the active site of this enzyme.
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PMID:NAD+ analogs in formation of new fluorophores on ultraviolet irradiation with D-glyceraldehyde-3-phosphate dehydrogenase. 710 Aug 95

The inactivation of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus and sturgeon by 3-chloroacetylpyridine--adenine dinucleotide, which is not active as a coenzyme, obeys pseudo-first-order kinetics. The mechanism of inactivation corresponds to an affinity label. Four moles of inactivator are incorporated per mole of tetramer for both enzymes. All the kinetic results strongly support the idea that neither enzyme exhibits negative cooperativity for the non-covalent binding of 3-chloroacetylpyridine--adenine dinucleotide. NAD+ strongly protects both glyceraldehyde-3-phosphate dehydrogenases from inactivation. In the presence of NAD+, the inactivation process seems also to exhibit pseudo-first-order kinetics. The fact that each kinetic experiment can be described by only one pseudo-first-order rate constant is unexpected. The results of thiol titration are in favor of the modification of the essential Cys-149, although another thiol group per monomer, probably Cys-153, becomes titratable on both native alkylated enzymes. This latter result suggests that the alkylation of the Cys-149 by the 3-chloroacetylpyridine--adenine dinucleotide should affect the tertiary structure of these enzymes.
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PMID:Affinity labeling of glyceraldehyde-3-phosphate dehydrogenase from sturgeon and Bacillus stearothermophilus by 3-chloroacetylpyridine--adenine dinucleotide. Kinetic studies. 717 94

The binding of NAD+ and of its fluorescent analogue, nicotinamide 1,N6-ethenoadenine dinucleotide, to glyceraldehyde-3-phosphate dehydrogenase purified from the muscles of young and old rats was studied in detail. Binding of the natural coenzyme was followed both by spectrophotometric titration of the extrinsic absorption band of the enzyme-NAD+ complex and from the degree of quenching of fluorescence of the protein. Binding of the coenzyme analogue was monitored by using the large enhancement in its fluorescence upon forming the complex with the enzyme. Both dinucleotides showed strong negative cooperativity in binding to the enzyme, similar to that displayed in their association with the rabbit muscle enzyme. The enzyme purified from old rats displayed a markedly reduced affinity toward the two dinucleotides, compared with the enzyme isolated from young animals. The various dissociation constants of both dinucleotides from the enzyme from young rats were remarkably similar to the corresponding constants in the rabbit muscle enzyme. The degree of negative cooperativity (i.e., the ratio between the dissociation constants from high- and low-affinity binding sites) in the "young" and "old" enzyme forms was not very different. It was concluded from these results that while modifications in the subunits take place upon aging, the intersubunit interaction is not significantly affected. Increasing concentrations of ATP were found to cause a gradual decrease in the negative cooperativity of NAD+ binding, which completely disappeared in the presence of 5 mM ATP. The observation that all four binding sites of the old enzyme display the same affinity toward NAD+ when the negative cooperativity is removed excludes the possibility that this enzyme form is a mixture of native and inactive species. The different dissociation constants of NAD+ from young and old enzyme forms in the presence of 5 mM ATP also demonstrate the occurrence of age-related modifications in the structure of the individual subunits.
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PMID:Age-related effects in coenzyme binding patterns of rat muscle glyceraldehyde-3-phosphate dehydrogenase. 730 93

The binding of nicotinamide--adenine dinucleotide (NAD+), nicotinamide--1,N6-ethenoadenine dinucleotide (epsilon NAD+), acetylpyridine--adenine dinucleotide (AcPyAD+), ATP, and adenosine diphosphoribose (ADP-ribose) to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (the enzyme) was examined. NAD+ and epsilon NAD+ were found to bind to the apoenzyme in a negatively cooperative manner, whereas AcPyAD+, ATP, and ADP-ribose bind non-cooperatively to the NAD+ sites. The strong negative cooperativity in coenzyme binding was found to be abolished in the presence of AcPyAD+ and strongly weakened by ATP, ADP, and AMP, but was not affected by the addition of ADP-ribose. These findings demonstrate that the mechanism of the negative cooperativity in coenzyme binding to the enzyme involves ligand-induced conformational changes between neighboring sites. These findings cannot be accounted for by the pre-existent asymmetry model. The results support our previous hypothesis that the structure of the pyridine moiety of the coenzyme analogues plays a role in orienting the adenine moiety in the adenine subsite, and thus affects the cooperativity observed in the binding of the coenzyme analogue.
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PMID:The sequential nature of the negative cooperativity in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. 744 64

Incorporation of L-[35S]cysteine into rabbit muscle glyceraldehyde-3-phosphate dehydrogenase was detected following incubation of the enzyme in a mixture containing glyceraldehyde-3-phosphate, NAD+ and the labeled cysteine. Insignificant binding occurred in the absence of either the substrate or NAD+, suggesting that formation of an acylated enzyme form was a prerequisite for the binding. Stoichiometry of the binding depended on the number of functioning active centers; up to 4 moles of L-[35S]cysteine bound per mole tetramer with fresh enzyme preparations. The L-[35S]cysteine incorporation depended on pH and was maximal when a group having pKa of 8.5 is protonated. To clarify the relevance of this finding to the effect of SH-containing compounds previously shown to decrease the rate of 3-phosphoglyceroyl-enzyme hydrolysis [Kuzminskaya et al., FEBS Lett. 336 (1993) 208-210], the pH-dependence of the effect of glutathione on the hydrolysis rate was determined and found to be close to the pH-dependence of L-[35S]cysteine binding.
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PMID:Interaction of glyceraldehyde-3-phosphate dehydrogenase with SH-containing compounds: evidence for the binding of L-cysteine and for the dependence of the binding on the functional state of the enzyme. 749 71


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