Chemical Nature of the Catalytic Sites in Glyceraldehyde-3-Phosphate Dehydrogenase

Harris, Ieuan; Meriwether, Blanche P.; Park, Jane Harting

doi:10.1038/198154a0

Cited by 239 publications

(60 citation statements)

References 20 publications

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“…However, it is possible to estimate this coenzyme binding affinity by performing simultaneous fluorimetric and spectrophotometric titrations of the appearance of NADH following successive additions of NAD' to a mixture of apoenzyme and glyceraldehyde 3-phosphate. The observed absorbance ( A ) and fluorescence (fl of NADH produced in such a reaction mixture can be written respectively as follows : (2) ' Owing to the presence of high concentrations of enzyme in the measuring cell the absorption spectrum of the dihydronicotinamide moiety of the NADH . acylenzyme complex is clearly distorted at wavelengths below 320 nm.…”

Section: Evaluation Of the Binding Aj'nity Of' The Acylenzyme For N Amentioning

confidence: 99%

Specific Interactions of 3-Phosphoglyceroyl-glyceraldehyde-3-Phosphate Dehydrogenase with Coenzymes

et al. 1976

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The binding of oxidized and reduced coenzyme (NAD ' and NADH) to 3-phosphoglyceroylglyceraldehyde-3-phosphate dehydrogenase has been studied spectrophotometrically and fluorimetrically. The binding of NAD+ to the acylated sturgeon enzyme is characterized by a significant quenching of the enzyme fluorescence (about 25 '%;) and the induction of a difference spectrum in the ultraviolet absorbance region of the enzyme. Both of these spectroscopic properties are quantitatively distinguishable from those of the corresponding binary enzyme . NAD ' complex. Binding isotherms estimated by gel filtration of the acylated enzyme are in close agreement to those obtained by spectrophotometric and fluorimetric titrations. Up to four NAD' molecules are bound to the enzyme tetramer. No anticooperativity can be detected in the binding of oxidized coenzyme, which is well described on the basis of a single class of four binding sites with a dissociation constant of 25 pM at 10 "C, pH 7.0.The binding of NADH to the acylenzyme has been characterized spectrophotometrically. The absorption band of the dihydronicotinamide moiety of the coenzyme is blue-shifted to 335 nm with respect to free NADH. In addition, a large hypochromicity (23%) is observed together with a significant increase of the bandwidth at half height of this absorption band. This last property is specific to the acylenzyme . NADH complex, since it disappears upon arsenolysis of the acylenzyme.The binding affinity of NADH to the acylated enzyme has been estimated by performing simultaneous spectrophotometric and fluorimetric titrations of the NADH appearance upon addition of NAD' to a mixture of enzyme and excess glyceraldehyde 3-phosphate. In contrast to NAD', the reduced COenzyme NADH appears to be relatively strongly bound to the acylated enzyme, the dissociation constant of the acylenzyme . NADH complex being estimated as 2.0 pM at 25 "C. In addition a large quenching of the NADH fluorescence (about 83 yo) is observed. The comparison of the dissociation constants of the coenzyme . acylenzyme complexes and the corresponding Michaelis constants suggests a reaction mechanism of the enzyme in which significant formation and dissociation of NAD' . acylenzyme and NADH . acylenzyme complexes occur. Under physiological conditions the activity of the enzyme can be regulated by the ratio of oxidized and reduced coenzymes.Possible reasons for the lack of anticooperativity in coenzyme binding to the acylated form of the enzyme are discussed.

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Section: Evaluation Of the Binding Aj'nity Of' The Acylenzyme For N Amentioning

confidence: 99%

Specific Interactions of 3-Phosphoglyceroyl-glyceraldehyde-3-Phosphate Dehydrogenase with Coenzymes

et al. 1976

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“…Similarities are indicated from comparisons without knowledge of enzyme structures (1)(2)(3) and from comparisons of short regions of glyceraldehyde phosphate dehydrogenase (GPDH), liver alcohol dehydrogenase (LADH), yeast alcohol dehydrogenase (YADH), lactate dehydrogenase (LDH), or glutamate dehydrogenase (GDH) (4)(5)(6)(7)(8)(9)(10). Long segments of possible but distant homology are found between GPDH and LADH (11) or GDH (12).…”

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confidence: 99%

Partial Similarities Between Yeast and Liver Alcohol Dehydrogenases

Jörnvall

1973

Proc. Natl. Acad. Sci. U.S.A.

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The primary structure of about half of the protein chain of yeast alcohol dehydrogenase has been determined and compared with the amino-acid sequences of other dehydrogenases. The enzyme is found to be distantly related to horse-liver alcohol dehydrogenase, although these two proteins have different quaternary structures and subunit sizes. Some regions show no significant similarities, but long segments within the N-terminal parts of the molecules are homologous, suggesting a common and important function for these segments. Ancestral connections between some different dehydrogenases can be concluded and the degree of evolutionary changes may be estimated.

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“…The tryptic peptide containing this cysteine has been isolated from several different lactate dehydrogenases, including both M4 and H4 isozymes, and in all cases the sequence has been closely conserved (15,16). If a single deletion is assumed to have occurred in the lactate dehydrogenase sequence, a reasonable comparison can be made of the sequence of this peptide with the sequence sumrounding the "essential" cysteine in yeast alcohol dehydrogenase (27), horse-liver alcohol dehydrogenase (27,28), and glyceraldehyde-3-phosphate dehydrogenase (29,30) (Fig. 4).…”

Section: Methodsmentioning

confidence: 99%

Aminoacid Sequence of Dogfish M ₄ Lactate Dehydrogenase

Taylor

Oxley

Allison

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

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About 80% of the aminoacid sequence of dogfish (Squalus acanthius) M4 lactate dehydrogenase (EC 1.1.1.27) has been elucidated. Several sequence homologies with peptides from pig H4 and pig M4 lactate dehydrogenase are identified. Histidine 195 is homologous to the essential histidine residue in pig H4 lactate dehydrogenase. Similarities in the sequence around the "essential" cysteine residue of lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and yeast and liver alcohol dehydrogenases are delineated.

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Chemical Nature of the Catalytic Sites in Glyceraldehyde-3-Phosphate Dehydrogenase

Cited by 239 publications

References 20 publications

Specific Interactions of 3-Phosphoglyceroyl-glyceraldehyde-3-Phosphate Dehydrogenase with Coenzymes

Specific Interactions of 3-Phosphoglyceroyl-glyceraldehyde-3-Phosphate Dehydrogenase with Coenzymes

Partial Similarities Between Yeast and Liver Alcohol Dehydrogenases

Aminoacid Sequence of Dogfish M ₄ Lactate Dehydrogenase

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Chemical Nature of the Catalytic Sites in Glyceraldehyde-3-Phosphate Dehydrogenase

Cited by 239 publications

References 20 publications

Specific Interactions of 3-Phosphoglyceroyl-glyceraldehyde-3-Phosphate Dehydrogenase with Coenzymes

Specific Interactions of 3-Phosphoglyceroyl-glyceraldehyde-3-Phosphate Dehydrogenase with Coenzymes

Partial Similarities Between Yeast and Liver Alcohol Dehydrogenases

Aminoacid Sequence of Dogfish M 4 Lactate Dehydrogenase

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Aminoacid Sequence of Dogfish M ₄ Lactate Dehydrogenase