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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)

The kinetic method and selective chemical modification have been used in studies of the kinetic manifestations of active site interactions in D-glyceraldehyde-3-phosphate dehydrogenase (GAP dehydrogenase). The reactions of glyceraldehyde and glyceraldehyde-3-phosphate oxidation were studied in the absence of substrate excess. In support of the data obtained previously it was shown that only a part of the tightly bound NAD molecules can be reduced after substrate addition. "Partial reducibility" is observed at various degrees of saturation of the enzyme with NAD involving a single NAD molecule per tetrametric enzyme. These facts can hardly be explained by assumption of functional non-equivalence of active sites, whether induced by coenzyme or preexisting in the apoenzyme. It was proven by selective alkylation of the catalytic SH groups that "partial reducibility" is due to the circumstance that equilibrium in the system under investigation is established at nearly equal NAD and NADH concentrations. A plot of initial reaction rates versus NAD concentration (at non-saturating substrate concentrations) gives S-shaped curves; this is explained by considerable enzyme activation upon saturation of the fourth site with coenzyme. After modification of three active sites with iodoacetate the S-shape of the curve disappeared. This fact leads to the conclusion that active site interactions are required for formation of the S-shaped curves. The activity of a single site functioning in the modified enzyme reached values equal to those of the active sites in the native enzyme in the fully activated state. A model is proposed which can explaine the variations in mode of enzyme activation in the native and modified states. It is suggested that the surroundings of all four SH groups must be altered in order to activate the enzyme; such changes can be induced either by alkylation of the SH groups or by NAD binding. Evidence is presented that important functional properties of GAP dehydrogenase cannot be elucidated at low enzyme concentrations and with excess of substrates: three active sites are saturated under such conditons and practically inactive, and the fourth site obeys Michaelis - Menten kinetics.
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PMID:[Kinetic manifestations of the interaction of active centers in swine skeletal muscle D-glyceraldehyde-3-phosphate dehydrogenase]. 18 4

A three-step procedure including affinity chromatography on NAD+-azobenzamidopropyl-Sepharose has been designed for the purification of yeast glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12] with maximized specific activity and maximized homogeneity with respect to affinity for the coenzyme, NAD+. Binding isotherms allow the analysis of cooperativity patterns that disclose both the average ligand affinity in the system and the distribution of ligands among the sites, only for systems with complete affinity homogeneity. The presence of affinity heterogeneity, resulting from multiple oligomeric species differing only in their affinity for coenzyme, gives rise to isotherms which falsely manifest apparent negative cooperativity. A method for distinguishing negative homotropic cooperativity from affinity heterogeneity is suggested.
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PMID:Negative homotropic cooperativity and affinity heterogeneity: preparation of yeast glyceraldehyde-3-phosphate dehydrogenase with maximal affinity homogeneity. 18 79

Binding of four molecules of NAD to pig muscle glyceraldehyde-3-phosphate dehydrogenase decreases the apparent reactivity of Cys-153 -- a residue exposed only temporarily -- towards PMB in all four subunits of the enzyme. However, the change of reactivity is not a linear function of the degree of saturation with coenzyme, inasmuch as the first two, tightly bound NAD's exert a much larger effect than do the other two. The apparent reactivity of Cys-153 was investigated in GAPD's produced by hybridization of enzymes modified on residue Cys-149 with different reagents. The homotetramer-NAD complexes of these modified species were shown to exhibit different dissociation constants: native GAPD less than (alkylated--Cys-149)-GAPD less than (mercaptidated--Cys-149)-GAPD. The tight binding of 2 NAD's on the hybrid tetramers decreases the reactivity to the same extent in liganded and non-liganded subunits. The decrease in the apparent reactivity of Cys-153 is due to the restriction of local conformational motility around thes residue. Binding of NAD shifts the equilibrium towards a more closed form of the protein, whereas the rate constant of mercaptide formation itself remains unaltered. These findings suggest that the NAD-induced conformational changes are also reflected in the local fluctuation of the protein around Cys-153. The subunit interactions still operate in the hybrids and mediate the NAD-induced conformational changes.
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PMID:Conformational motility in D-glyceraldehyde-3-phosphate dehydrogenase influenced by subunit interactions. 18 92

The NAD-binding of pig muscle glyceraldehyde-3-phosphate dehydrogenase after modifying certain SH-groups of the enzyme was studied by spectrophotometric titration and gel-chromatography. The enzyme modified on residue Cys-149 with N-ethylmaleimide, iodoacetate or p-mercuribenzoate binds NAD less tightly than does the unmodified enzyme. However, the differences between the NAD-binding sites characteristic of the native tetrameric enzyme are largely retained in the modified enzyme. Residue Cys-153 is near to the surface of the enzyme and is temporarily exposed due to the fluctuation of the protein. It can be modified only after blocking Cys-149. Modification of residue Cys-153 with N-ethylmaleimide does not further influence NAD-binding. Reaction of Cys-153 with p-mercuribenzoate does not directly cause the release of NAD. In this case the enzyme-coenzyme complex decomposes due to secondary conformational changes. This followed from the finding that the disappearance of the absorption band characteristic of the enzyme-coenzyme charge transfer complex is a first order process is equal to that of the structural changes following mercaptide formation of Cys-153.
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PMID:Effect of modification of SH-groups in D-glyceraldehyde-3-phosphate dehydrogenase on the properties of enzyme--coenzyme complex. 18 95

The effect of NAD on the binding of 1-anilino-8-naphthalene sulfonate (ANS) to yeast glyceraldehyde-3-phosphate dehydrogenase has been studied using difference spectrophotometric and fluorescence techniques. Coenzyme addition causes the displacement of ANS from its complex with the dehydrogenase, as suggested by the effect of NAD on the fluorescence of the enzyme--ANS complex, as well as on the magnitude of the difference spectrum of the complex. Adenine containing NAD fragments, adenosine, 5'-AMP, and ADP were shown to compete with ANS for the common site on the enzyme using fluorimetric technique; in the case of adenosine and 5'-AMP a direct method of analytical ultracentrifugation was also employed. The results obtained by both methods suggest the dye binding at the adenine subsite of the dehydrogenase. The interaction with ANS causes no detectable conformational changes of the protein. The fluorescence of the dye-enzyme complex increases and the emission maximum shifts to shorter wavelengths on addition of nicotinamide mononucleotide. This suggest some conformational changes to occur in the microenvironment of the bound dye in response to the interaction with the ligand in the nicotinamide subsite. The participation of the nicotinamide subsite of the active center in determining the character of conformational transitions associated with coenzyme binding to glyceraldehyde-3-phosphate dehydrogenase is discussed.
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PMID:[Use of a fluorescent probe for the study of the active center of D-glyceraldehyde-3-phosphate dehydrogenase]. 19 46

The structure of the active center of glyceraldehyde-3-phosphate dehydrogenase and the arrangement of subunits in the tetrameric molecule is delineated. The mechanism of cooperative effects in the oligomer is considered, and the involvement of various regions of the active center and of different-subunit contact area in the realization of the cooperative phenomena is discussed. A special attention is paid to the effect of NAD+ bound to one of the subunits of the tetramer on the structure of an adjacent subunit and to the problem of the participation of the coenzyme in the creation of anion-binding sites of the enzyme. The conditions of reversible dissociation of the tetrameric apoenzyme molecule into dimers are depicted, and the role of NAD+ in the organization of the quaternary structure of the dehydrogenase is discussed. The problem of catalytic activity of the dimeric form of the enzyme is argued.
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PMID:[Cooperative properties of D-glyceraldehyde-3-phosphate dehydrogenase]. 19 81

[omega-(3-Acetylpyridinio)-n-alkyl]adenosine pyrophosphates are coenzyme analogs of NAD. The adenosine pyrophosphate moiety and the 3-acetylpyridine ring of the analogs are connected by n-alkyl chains of different lengths (ethyl--hexyl). The analogs form strong dissociating complexes with lactate dehydrogenase. The complex formation is predominantly achieved by interaction of the ADP moiety with its respective binding domain at the active site. The redox potentials of the analogs and NAD are of similar magnitude. The coenzyme function of the analogs depends upon the length of the hydrocarbon chain. Lactate dehydrogenase and alcohol dehydrogenases from yeast and horse liver do not catalize hydrogen transfer from their substrates to any other alkyl analog but [4-(3-acetylpyridinio)-n-butyl]adenosine pyrophosphate, aldehyde dehydrogenase from horse liver catalizes hydrogen transfer from acetaldehyde to the pentyl derivative and glyceraldehyde-3-phosphate dehydrogenase catalizes hydrogen transfer to both analogs. In no case, hydrogen transfer from or to one of the 3-acetylpyridine-n-alkyl analogs proceeded with a velocity comparable to NAD or its 3-acetylpyridine analog. The results show that the nicotinamide bound ribose in NAD is involved in the binding and the activation of the coenzyme.
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PMID:[The properties of [omega(3-acetylpyridinio)-n-alkyl]adenosine pyrophosphates, structural analogs of the coenzyme NAD (author's transl)]. 19 87

The effect of borate on glyceraldehyde-3-phosphate dehydrogenase from human, pig and rabbit muscle was studied. At lower concentration of borate only the dehydrogenase activity is inhibited, reversibly and competitively against NAD. At concentration of borate above 6 mM the plots of 1/v versus borate concentration become nonlinear and the inhibition is extended to the esterase and acetylphosphatase activities. In certain conditions a time-dependent inactivation and reactivation was observed. The direct interaction between borate (if present at concentration of at least 6 mM) and glyceraldehyde-3-phosphate dehydrogenase is postulated, the possible site of the reaction being the histidine residue(s). The esterase activity of the human muscle enzyme and the effect of borate on it are different from the other mammalian enzymes.
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PMID:Effect of borate on the catalytic activities of muscle glyceraldehyde-3-phosphate dehydrogenase. 20 Apr 28

The stabilizing effect of the coenzyme (NAD) on the structure of glyceraldehyde-3-phosphate dehydrogenase from lamprey and porcine muscles with respect to proteolysis and heat denaturation was studied. The process of heat denaturation was followed by the changes in specific activity of the enzymes; that of proteolysis--by the changes in specific activity and circular dichroism. It was shown that in both cases NAD at saturating concentration exerts a far weaker stabilizing effect on the structure of glyceraldehyde-3-phosphate dehydrogenase from lamprey muscle than on that of the porcine muscle enzyme. The coensyme-dependent stabilization of lamprey muscle glyceraldehyde-3-phosphate dehydrogenase does not differ from that of mammalian muscle enzyme. Possible interrelationship between the phenomenon observed and the molecular mechanism of thermal adaptation in the cold-blooded animals is discussed.
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PMID:[Effect of coenzyme on conformational stability of glyceraldehyde-3-phosphate dehydrogenase from muscles of ecto- and endothermic animals]. 20 5

An NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC. 1.2.1.12) has been purified from spinach leaves as a homogeneous protein of 150,000 daltons. Kinetic constants of 2.5 . 10(-4) M and 4 . 10(-4) M have been calculated for NAD+ and glyceraldehyde-3-phosphate, respectively. The amino acid composition is characterized by a cysteine content higher than that found in analogous enzymes. On sodium dodecyl sulphate gel electrophoresis, the native enzyme dissociates into two subunits of 37,000 and 14,000 daltons. The two subunits have been isolated in equimolar amounts by gel filtration; end-group analysis shows that alanine is the N-terminal residue of the large subunit, while serine is found at the N-terminus of the small subunit. Comparison of amino acid analysies and peptide maps shows that the two subunits have a different amino acid sequence. These results indicate that the NAD+-dependent glyceraldehyde-3-phosphate, dehydrogenase, isolated from spinach leaves has an atypical oligomeric structure, the protomer being formed by two different subunits.
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PMID:Purification and properties of NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from spinach leaves. 20 24


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