Mechanism of Action of Glyceraldehyde-3-Phosphate Dehydrogenase

Racker, Efraim; Krimsky, I.

doi:10.1038/1691043a0

Cited by 69 publications

(43 citation statements)

References 12 publications

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“…Turbid (NH4)2SO4 suspensions developed crystals over a period of months, which were bipyramidal and grew up to 1 mm in size. The crystals were yellow, as was the concentrated eluate from the column, indicative of a normal Racker band (Racker & Krimsky, 1952). The specific activity of the purified enzyme was 180-200 units/mg, comparable with the highest values for this enzyme from other sources.…”

Section: General Behaviour Of the Enzymes On Dye-ligand Columnsmentioning

confidence: 60%

“…The Z. mobilis glyceraldehyde-phosphate dehydrogenase subunit appears to be about 10% larger than usual, but the number (four) in the active enzyme is normal (Harris & Waters, 1976). Moreover it has characteristic properties, including tightly bound NAD+ (evidenced by the yellow colour; Racker & Krimsky, 1952) and highly sensitive thiol groups, which can be reversibly oxidized. Phosphoglycerate kinase is normal structurally and kinetically, except for the lack of sulphate activation which occurs with most other phosphoglycerate kinases (Fifis & Scopes, 1978).…”

Section: Discussionmentioning

confidence: 99%

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Isolation and properties of the glycolytic enzymes from Zymomonas mobilis. The five enzymes from glyceraldehyde-3-phosphate dehydrogenase through to pyruvate kinase

Pawluk¹,

Scopes²,

Griffiths-Smith³

1986

Biochemical Journal

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The five glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase were each purified from extracts of Zymomonas mobilis cells, by using dye-ligand chromatography as the principal step. Two procedures, producing three and two of the enzymes respectively, are described in detail. Z. mobilis glyceraldehyde-phosphate dehydrogenase was found to be similar in most respects to the enzyme from other sources, except for having a slightly larger subunit size. Phosphoglycerate kinase has properties typical for this enzyme; however, it did not show the sulphate activation effects characteristic of this enzyme from most other sources. Phosphoglycerate mutase is a dimer, partially independent of 2,3-bisphosphoglycerate, and has a high specific activity. Enolase was found to be octameric; otherwise its properties were very similar to those of the yeast enzyme. Pyruvate kinase is unusual in being dimeric, and not requiring K+ for activity. It is not allosterically activated by sugar phosphates, having a high activity in the absence of any effectors. Some quantitative differences in the relative amounts of these enzymes, compared with eukaryotic species, are ascribed to the fact that Z. mobilis utilizes the Entner-Doudoroff pathway rather than the more common Embden-Meyerhoff glycolytic route.

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Section: General Behaviour Of the Enzymes On Dye-ligand Columnsmentioning

confidence: 60%

Section: Discussionmentioning

confidence: 99%

Isolation and properties of the glycolytic enzymes from Zymomonas mobilis. The five enzymes from glyceraldehyde-3-phosphate dehydrogenase through to pyruvate kinase

Pawluk¹,

Scopes²,

Griffiths-Smith³

1986

Biochemical Journal

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“…2), thereby resembling the absorption spectra of MFF [2] and of methanol dehydrogenase (N. Arfman et al, unpublished results), but not that of (rabbit muscle) glyceraldehyde-3-phosphate dehydrogenase (which also contains tightly bound NAD) which has an absorption maximum at 360nm [24]. Samples of enzyme denaturated by heating were inactive in the pyrroloquinoline quinone assay and also flavins were absent, as judged from the absorption spectrum.…”

Section: Purification and Characterization Of Ndma-adhmentioning

confidence: 98%

Nicotinoprotein [NAD(P)‐containing] alcohol/aldehyde oxidoreductases

Ophem

Beeumen

Duine

1993

European Journal of Biochemistry

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Extracts of Gram-positive bacteria like Rhodococcus rhodochrous, Rhodococcus erythropolisand Amycolatopsis methanolica, but not those of several Gram-negative ones, showed dehydrogenase activity for ethanol as well as for methanol when 4-nitroso-N, N-dimethylaniline (NDMA) was used as electron acceptor. Chromatography of extracts of the first two organisms revealed one activity for both substrates, that of A. methanolica two activities, one of which is able to oxidize methanol and has been purified (Bystrykh, L. V., Govorukhina, N. I., van Ophem, P. W., Hektor, H. J., Dijkhuizen, L. and Duine, J. A., unpublished results). The other, indicated as NDMA-dependent alcohol dehydrogenase (NDMA-ADH), was purified to homogeneity. It is a trimeric enzyme consisting of subunits of 39 kDa and one firmly bound NAD as cofactor. Although NDMA-ADH shows structural similarity with the long-chain, zinc-containing, NAD(P)-dependent alcohol dehydrogenases with respect to the N-terminal sequence up to residue 41 (56% identity with horse liver alcohol dehydrogenase), the enzymes are catalytically different since NDMA-ADH is unable to use NAD(P)(H) as a coenzyme and NAD(P)-dependent alcohol dehydrogenases are inactive with NDMA (in the absence of NAD). Comparison of the NDMA-ADH properties with those of the methanol-oxidizing enzyme of A. methanolica, Mycobacterium gastri and Bacillus methanolica C1, and formaldehyde dismutase of Pseudomonas putida F61 revealed large differences in structural as well as catalytic properties, in spite of the fact that all are nicotinoproteins [enzymes which have bound NAD(P) as a cofactor]. It is concluded, therefore, that NDMA-ADH is a novel type of nicotinoprotein alcohol dehydrogenase.In the search for a methanol-oxidizing activity in Amycolatopsis methanolica, an assay able to detect alcohol dehydrogenases having NAD(P) as cofactor was applied, using 4-nitroso-N, N-dimethylaniline (NDMA) as electron acceptor [ 1). However, upon applying anion-exchange chromatography on extracts exhibiting such a property, two separate activities were found in the eluted fractions, one showing activity with alcohols including methanol, the other excluding methanol. The enzyme responsible for the first activity [the Correspondence to J. A. Duine,

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“…In the presence of NAD, the kinetics were followed at 370nm by measuring the disappearance of the broad absorption band characteristic of the enzymecoenzyme complex [16]. It was observed earlier that the disappearence of the broad absorption band of the holoenzyme on alkylation with iodoacetamide is followed by the formation of a new band with a sharp maximum at 325 nm [17].…”

Section: Kinetics Of Alkylationmentioning

confidence: 99%

Ion‐Pair Formation as a Source of Enhanced Reactivity of the Essential Thiol Group of d‐Glyceraldehyde‐3‐Phosphate Dehydrogenase

Polgár

1975

European Journal of Biochemistry

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The reactivity and the mode of activation of the essential -SH group (Cys-149) of D-glyceraldehyde-3-phosphate dehydrogenase have been studied by means of a spectrophotometric method [Polgar, L., FEBS Lett. 38, 187-190 (1974)], capable of detecting the dissociated form of the thiol group in proteins.Alkylations of Cys-149 of NAD-free ~-glyceraldehyde-3-phosphate dehydrogenase with iodoacetamide and iodoacetate were investigated. The corrected absorbance change on alkylation at 250 nm (which is a direct parameter of the dissociation of the thiol group) and the alkylation rate were determined as a function of pH. The pH profiles of both dissociation and alkylation rate of Cys-149 conform to doubly sigmoid curves. All these curves implicate two ionizing groups (pK, = 5.5, pK2 = 8.2).It is concluded that there are two reactive forms of the -SH group in the apoenzyme between pH 5 and 10. One reactive form corresponds to the free mercaptide ion. The other can be identified with an ion-pair composed of a mercaptide ion and some base, possibly the imidazolium group of His-176.The ion-pair has lower molar absorption coefficient and nucleophilicity than the free mercaptide ion.The two reactive forms are transformed into each other with pK2 = 8.2. The ion-pair decomposes to a nondissociated thiol group and a protonated base with pK, = 5.5.In the presence of NAD, only the pH-rate profile of alkylation of ~-glyceraldehyde-3-phosphate dehydrogenase was measured (at 370 nm). Using iodoacetamide as alkylating agent we also obtained a doubly sigmoid curve. A slight downward shift on pK, and an upward shift in pK2 indicate that the ion-pair exists in a somewhat wider pH-range in the enzyme-coenzyme complex.An increase in the ionic strength of the reaction mixture from 0.09 to 0.45 M does not abolish the doubly sigmoid character of the curves determined either in the presence or in the absence of NAD.Previous studies have shown that the -SH group of thiolsubtilisin [1,2] and papain [3] forms an ionpair with the imidazole group of a neighboring histidine residue. At slightly acidic pH this mercaptideimidazolium ion-pair may account for the high reactivity as compared to what is expected from the normal dissociation of an -SH group. Thus the pHdependence of alkylation of papain with haloacetamides displays a doubly sigmoid curve with parameters corresponding to the reactivity of the free Enzyme. D-Glyceraldehyde 3-phosphate : NAD oxidoreductase (phosphorylating) (EC 1.2

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Mechanism of Action of Glyceraldehyde-3-Phosphate Dehydrogenase

Cited by 69 publications

References 12 publications

Isolation and properties of the glycolytic enzymes from Zymomonas mobilis. The five enzymes from glyceraldehyde-3-phosphate dehydrogenase through to pyruvate kinase

Isolation and properties of the glycolytic enzymes from Zymomonas mobilis. The five enzymes from glyceraldehyde-3-phosphate dehydrogenase through to pyruvate kinase

Nicotinoprotein [NAD(P)‐containing] alcohol/aldehyde oxidoreductases

Ion‐Pair Formation as a Source of Enhanced Reactivity of the Essential Thiol Group of d‐Glyceraldehyde‐3‐Phosphate Dehydrogenase

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