On the Mechanism of Thiamine Action

White, Fred G.; Ingraham, Lloyd L.

doi:10.1021/ja01500a077

Cited by 24 publications

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“…Hydrolysis of Acetyl-TPP. The results of the hydrolysis of acetyl-TPP showing lability at neutral pH and relative stability under acidic conditions is a general feature expected of the compound and similar to results seen with model compounds (Breslow & McNelis, 1960; White & Ingraham, 1960Daigo & Reed, 1962;Lienhard, 1966). However, preliminary results of a more detailed kinetic analysis of the hydrolysis reaction have shown that significant differences exist between the model compound, 2-acetyl-3,4-dimethylthiazolium ion (Lienhard, 1966), and acetyl-TPP.2 These differences appear to be a direct result of the presence of the pyrimidine ring and will be presented in a future paper along with mechanistic details of the hydration and adduct formation reactions.…”

Section: Discussionsupporting

confidence: 78%

Synthesis and properties of 2-acetylthiamin pyrophosphate: an enzymatic reaction intermediate

Gruys¹,

Halkides²,

Frey³

1987

Biochemistry

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The synthesis of 2-acetylthiamin pyrophosphate (acetyl-TPP) is described. The synthesis of this compound is accomplished at 23 degrees C by the oxidation of 2-(1-hydroxyethyl)thiamin pyrophosphate using aqueous chromic acid as the oxidizing agent under conditions where Cr(III) coordination to the pyrophosphoryl moiety and hydrolysis of both the pyrophosphate and acetyl moieties were prevented. Although the chemical properties exhibited by acetyl-TPP are similar to those of the 2-acetyl-3,4-dimethylthiazolium ion examined by Lienhard [Lienhard, G.E. (1966) J. Am. Chem. Soc. 88, 5642-5649], significant differences exist because of the pyrimidine ring in acetyl-TPP. Characterization of acetyl-TPP by ultraviolet spectroscopy, 1H NMR, 13C NMR, and 31P NMR provided evidence that the compound in aqueous solution exists as an equilibrium mixture of keto, hydrate, and intramolecular carbinolamine forms. The equilibria for the hydration and carbinolamine formation reactions at pD 1.3 as determined by 1H NMR are strongly dependent on the temperature, showing an increase in the hydrate and carbinolamine forms at the expense of the keto form with decreasing temperature. The concentration of keto form also decreases with increasing pH. Acetyl-TPP is stable in aqueous acid but rapidly deacetylates at higher pH to form acetate and thiamin pyrophosphate. Trapping of the acetyl moiety in aqueous solution occurs efficiently with 1.0 M hydroxylamine at pH 5.5-6.5 to form acetohydroxamic acid and to a much smaller extent with 1.0 M 2-mercaptoethanol at pH 4.0 and 5.0 to form thio ester. Transfer of the acetyl group to 0.5 M dihydrolipoic acid at pH 5.0 and 1.0 M phosphate dianion at pH 7.0 is not observed to any significant extent in water. The kinetic and thermodynamic reactivity of acetyl-TPP with water and other nucleophiles is compatible with a hypothetical role for acyl-TPPs as enzymatic acyl-transfer intermediates.

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Section: Discussionsupporting

confidence: 78%

Synthesis and properties of 2-acetylthiamin pyrophosphate: an enzymatic reaction intermediate

Gruys¹,

Halkides²,

Frey³

1987

Biochemistry

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“…It should be noted that reaction e is presumed to be reversible. It is assumed that the presence of the enzyme stabilizes the acetyl-DPT complex since acyl-thiazolium compounds are extremely unstable in water or phosphate (Breslow and McNelis, 1960;White and Ingraham, 1960). However, the inability to demonstrate acetate exchange in a variety of conditions suggests that the equilibrium of reaction e greatly favors the formation of acetyl-CoA (or acetyl phosphate as discussed below).…”

Section: Discussionmentioning

confidence: 99%

DEGRADATION OF PYRUVATE BY MICROCOCCUS LACTILYTICUS III

Whiteley

McCormick

1963

J Bacteriol

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AND N. G. MCCORMICK. Degradation of pyruvate by Micrococcus lactilyticus. III. Properties and cofactor requirements of the carbon dioxide-exchange reaction. J. Bacteriol. 85:382-393. 1963.-At an acid pH, extracts of Micrococcus lactilyticus (Veillonella alcalescens) catalyze the oxidative decarboxylation of pyruvate to carbon dioxide, hydrogen, and acetyl phosphate, and the rapid exchange of carbon dioxide into the carboxvl group of pyruvate. These reactions take place only under anaerobic conditions and require phosphate (or arsenate), a reducing agent, diphosphothiamine, coenzyme A, an electron acceptor (ferredoxin, flavins, dyes, or certain inorganic anions), and a divalent cation (Co++ > Mni > Mg+ > Fe++). High concentrations of coenzyme A and electron acceptors stimulate pyruvate breakdown but inhibit CO2 exchange. Exchange is also inhibited by p-chloromercuribenzoate but not by arsenite. Extracts rapidly lose the ability to mediate the exchange reaction after passage through diethylaminoethylor triethylaminoethyl-cellulose or Dowex-1; this loss in activity may be prevented by adding a reducing agent and the above cofactors. The exchange of CO2 and formate by M. lactilyticus is compared. Strict anaerobes such as Clostridium butyricum (Wolfe and O'Kane, 1953), Micrococcus lactilyticus (Whiteley and Ordal, 1957), C. saccharobutyricum (Delavier-Klutchko, 1959), and Micrococcus LC (Peel, 1960) are able to degrade pyruvate to acetate, carbon dioxide, and hydrogen. This overall reaction (equation 1) requires phosphate and a slightly acid pH, and involves a pyruvate-acetate + CO2 + H2 (1)

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“…In the E. coli system, these are presumed to be competing reactions, since larger amounts of acetyl phosphate are produced at high concentrations of phosphate. The acyl thiazolium salts are extremely unstable (Breslow and MeNelis, 1960;White and Ingraham, 1960), and this may account for the low levels of acetate exchange observed with this system. However, in the 3ll.…”

Section: Discussionmentioning

confidence: 99%

DEGRADATION OF PYRUVATE BY MICROCOCCUS LACTILYTICUS I

1962

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At an alkaline pH, extracts of Micrococcus lactilyticus2 catalyze the phosphoroclastic degradation of pyruvate to formate and acetyl phosphate and the rapid exchange of formate into the carboxyl group of pyruvate. At an acid pH, hydrogen, carbon dioxide, and acetyl phosphate are produced, and carbon dioxide is exchanged into the carboxyl group of pyruvate. A concentration of approximately 1 M phosphate is required for the phosphoroclastic reaction and formate exchange; the production of carbon dioxide and hydrogen is greatly inhibited by high concentrations of phosphate. Formate exchange requires a divalent metal ion and is stimulated by reducing agents and an atmosphere of hydrogen. Inhibition by p-chloromercuribenzoate, ZnH, Cd+, and arsenite indicates that sulfhydryl groups on the enzyme are involved in the reaction; the inhibition by arsenite and Cd may be relieved by 2,3-dimercaptopropanol, suggesting that vicinal dithiols may be required. Inhibition by hypophosphite may reflect a competition with formate for a site on the enzyme. At an alkaline pH, oa-ketobutyrate is degraded to propionate and formate, whereas a-ketoglutarate is fermented to succinate, propionate, carbon dioxide, hydrogen, and formate. Formate is exchanged into the carboxyl groups of aketobutyrate and a-ketoglutarate under these conditions. Only traces of a-ketovalerate and

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On the Mechanism of Thiamine Action

Cited by 24 publications

References 0 publications

Synthesis and properties of 2-acetylthiamin pyrophosphate: an enzymatic reaction intermediate

Synthesis and properties of 2-acetylthiamin pyrophosphate: an enzymatic reaction intermediate

DEGRADATION OF PYRUVATE BY MICROCOCCUS LACTILYTICUS III

DEGRADATION OF PYRUVATE BY MICROCOCCUS LACTILYTICUS I

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