[15] α-Hydroxyethylthiamine diphosphate and α,β-dihydroxyethylthiamine diphosphate

Krampitz, Lester O.; Votaw, Robert G.

doi:10.1016/0076-6879(66)09019-0

Cited by 4 publications

(3 citation statements)

References 4 publications

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“…The concentration of [14C]TPP was determined spectrophotometrically at 267 nm in 20 mM potassium phosphate, pH 7.0 (c = 8550 M'1 cm'1) (Wittorf & Gubler, 1970). Product purity was checked by chromatography in 1-propanol/1 M ammonium formate (pH 5.0)/H2O (65:15:25) on Whatman 3MM paper (Krampitz & Votaw, 1966). Labeled compounds were visualized by autoradiography.…”

Section: Assay Of Dihydrolipoamide Acetyltransferase (E2) Andmentioning

confidence: 99%

Active-site modification of mammalian pyruvate dehydrogenase by pyridoxal 5'-phosphate

Stepp¹,

Reed²

1985

Biochemistry

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The pyruvate dehydrogenase multienzyme complex from bovine kidney and heart is inactivated by treatment with pyridoxal 5'-phosphate and sodium cyanide or sodium borohydride. The site of this inhibition is the pyruvate dehydrogenase (E1) component of the complex. Inactivation of E1 by the pyridoxal phosphate-cyanide treatment was prevented by thiamin pyrophosphate. Equilibrium binding studies showed that E1 contains two thiamin pyrophosphate binding sites per molecule (alpha 2 beta 2) and that modification of E1 increased the dissociation constant (Kd) for thiamin pyrophosphate about 5-fold. Incorporation of approximately 2.4 equiv of 14CN per mole of E1 tetramer in the presence of pyridoxal phosphate resulted in about a 90% loss of E1 activity. Radioactivity was incorporated predominantly into the E1 alpha subunit. Radioactive N6-pyridoxyllysine was identified in an acid hydrolysate of the E1-pyridoxal phosphate complex that had been reduced with NaB3H4. The data are interpreted to indicate that in the presence of sodium cyanide or sodium borohydride, pyridoxal phosphate reacts with a lysine residue at or near the thiamin pyrophosphate binding site of E1. This binding site is apparently located on the alpha subunit.

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Section: Assay Of Dihydrolipoamide Acetyltransferase (E2) Andmentioning

confidence: 99%

Active-site modification of mammalian pyruvate dehydrogenase by pyridoxal 5'-phosphate

Stepp¹,

Reed²

1985

Biochemistry

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“…The decarboxylated products (such as hydroxyethylidene-TPP (HE-TPP), Scheme 3) are central intermediates in all TPP-dependent reactions [3, 4]. …”

Section: Introductionmentioning

confidence: 99%

Radical reactions of thiamin pyrophosphate in 2-oxoacid oxidoreductases

Reed

Ragsdale

Mansoorabadi

2012

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Thiamin pyrophosphate (TPP) is essential in carbohydrate metabolism in all forms of life. TPP-dependent decarboxylation reactions of 2-oxo-acid substrates result in enamine adducts between the thiazolium moiety of the coenzyme and decarboxylated substrate. These central enamine intermediates experience different fates from protonation in pyruvate decarboxylase to oxidation by the 2-oxoacid dehydrogenase complexes, the pyruvate oxidases, and 2-oxoacid oxidoreductases. Virtually all of the TPP-dependent enzymes, including pyruvate decarboxylase, can be assayed by 1-electron redox reactions linked to ferricyanide. Oxidation of the enamines is thought to occur via a 2-electron process in the 2-oxoacid dehydrogenase complexes, wherein acyl group transfer is associated with reduction of the disulfide of the lipoamide moiety. However, discrete 1-electron steps occur in the oxidoreductases, where one or more [4Fe-4S] clusters mediate the electron transfer reactions to external electron acceptors. These radical intermediates can be detected in the absence of the acyl-group acceptor, coenzyme A (CoASH). The π-electron system of the thiazolium ring stabilizes the radical. The extensively delocalized character of the radical is evidenced by quantitative analysis of nuclear hyperfine splitting tensors as detected by electron paramagnetic resonance (EPR) spectroscopy and by electronic structure calculations. The second electron transfer step is markedly accelerated by the presence of CoASH. While details of the second electron transfer step and its facilitation by CoASH remain elusive, expected redox properties of potential intermediates limit possible scenarios.

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“…Thiochrome diphosphate was prepared according to the method of Nishimune et al (1988) while oxyThDP (in which the 4'-amino group is replaced by a hydroxyl) was a gift from Dr Stefan Konig (Martin-Luther-Universitat Halle-Wittenberg). he-ThDP was prepared by the method of Krampitz and Votaw (1966), separated from unreacted ThDP by ion-exchange chromatography (Deus et al, 1970), and freezedried. The product was completely free of ThDP, as shown by 'H-NMR ; he-ThDP concentrations were determined by absorbance measurements at 272.5 nm (Schellenberger and Hiibner, 1965).…”

Section: Preparation Of Apoenzyme All Glassware Plastic Cuvettesmentioning

confidence: 99%

The Role of His113 and His114 in Pyruvate Decarboxylase from Zymomonas Mobilis

Schenk

Leeper

England

et al. 1997

European Journal of Biochemistry

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Pyruvate decarboxylase (PDC) is one of several enzymes that require thiamin diphosphate (ThDP) and a divalent cation as essential cofactors. Recently, the three-dimensional structures of the enzyme from two yeasts have been determined. While these structures shed light on the binding of the cofactors and the reaction mechanism, the interactions between the substrate pyruvate and the enzyme remain unclear. We have used PDC from Zynzomonas mobilis as a model for these enzymes in order to study substrate binding. The recombinant enzyme was expressed in Escherichia coli. High yield, simplicity of purification, high stability and simple kinetics make this model well suited for these studies. Activity measurements in the pH range between 5.8 and 8.5 indicated that a His residue may be involved in substrate binding. Analysis of an alignment of all known PDC protein sequences showed two invariant His residues (His113 and Hisll4) which, according to the crystal structure of yeast PDC, are in the vicinity of the active site. Here we demonstrate that replacement of His114 by Gln does not have a great effect on cofactor and substrate binding. However, the k,,, is decreased indicating that Hisl 14 may assist in catalysis. In contrast, substitution of His113 by Gln renders the enzyme completely inactive. This mutant has decreased affinity for both cofactors, as revealed by measurements of tryptophan fluorescence quenching. However, this decreased affinity is insufficient to account for the complete loss of activity. Despite its inability to support overall catalysis, this [Glnl13]PDC mutant is capable of releasing acetaldehyde from 2-( 1 -hydroxyethyl)thiamin diphosphate supplied exogenously. It is proposed that upon substrate binding, His113 is placed close to C2 of the thiazole ring. Subsequent deprotonation of this atom leads to a conformational change that allows a flexible loop (residues 105-112) that precedes Hisl 13 to close over the active site. Hence, replacement of His113 by another residue interferes with this closure of the active site and thus disrupts the catalytic process.

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[15] α-Hydroxyethylthiamine diphosphate and α,β-dihydroxyethylthiamine diphosphate

Cited by 4 publications

References 4 publications

Active-site modification of mammalian pyruvate dehydrogenase by pyridoxal 5'-phosphate

Active-site modification of mammalian pyruvate dehydrogenase by pyridoxal 5'-phosphate

Radical reactions of thiamin pyrophosphate in 2-oxoacid oxidoreductases

The Role of His113 and His114 in Pyruvate Decarboxylase from Zymomonas Mobilis

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