- The reversible enolization and hydration of pyruvate: Possible roles of keto, enol, and hydrated pyruvate in lactate dehydrogenase catalysis
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The reversible enolization and hydration of pyruvic acid and pyruvate anion were monitored using spectrophotometric methods at several temperatures. Widely varying values for the equilibrium constant for the enolization of pyruvic acid and pyruvate ion appear in the literature. To accurately determine the position of equilibrium for the enolization reaction, we have developed a method that gives consistent results in which purified samples of sodium pyruvate are first "titrated" with triiodide ion to remove any triiodide-scavenging impurities such as those resulting from aldol condensation reactions. After reequilibration to allow the regeneration of enol pyruvate, the addition of small quantities of triiodide result in an initial burst in the decrease of absorbance at 353 nm, followed by the much slower zero-order decrease due to the formation of new enol pyvuvate molecules. The absorbance change during the burst phase of the reaction is proportional to the enol concentration plus that of any triiodide-scavenging impurity which may be present in the original pyruvate solution. Thus, as the quantity of triiodide used in the pretreatment stage of the experiments is increased, these burst absorbance changes, ΔA, decrease until a constant value of ΔA is reached. Accordingly, this final ΔA value is proportional to enol pyruvate (or enol pyruvic acid) in the absence of triiodide-scavenging impurity, allowing the accurate and reproducible determinations of Kenol. The equilibrium constants for both pyruvate and pyruvic acid are relatively temperature insensitive and, typically, Kenol (pyruvate anion) = 2.6 × 10-5 and Kenol (pyruvic acid) = 7.8 × 10-5 at 25.0°C. The zero-order phase of the reaction of triiodide ion may be used to calculate rate constants for enolization. The hydration and dehydration of pyruvic acid were followed directly by following absorbance changes in the peak at 340 nm due to the keto group. The thermodynamic and kinetic results reported in this paper are used to help determine whether the observed "substrate" inhibition of the lactate dehydrogenase catalyzed reduction of pyruvate is actually caused by keto, hydrated, or enol pyruvate.
- Esposito,Lukas,Meany,Pocker
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- A comparative study of the enolization of pyruvate and the reversible dehydration of pyruvate hydrate
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The enolization of pyruvate and the reversible dehydration of pyruvate hydrate were studied at 25.0°C using spectrophotometric methods. The enolization of pyruvate was followed at 353 nm by monitoring the rate of uptake of triiodide ion. The dehydration of pyruvate hydrate was initiated by introducing small quantities of preacidified solutions of pyruvic acid containing, at the kinetic zero, ca. 60% of the hydrate into buffer solutions. A decrease in absorbance at 325 nm took place as the reaction progressed to a final solution composition of 6% hydrate. The reactions were studied in acetate, MES, phosphate, arsenate, imidazole, 1-methylimidazole, HEPES, Tris, and borate buffers. The dehydration of pyruvate hydrate was found to be sensitive toward general-acid and general-base catalysis, while the enolization of pyruvate was catalyzed only by the basic components of the buffers studied. The corresponding rate coefficients were determined for the acidic and basic catalysts, and taking into account the appropriate statistical correction factors associated with the capacity of the catalysts to donate and accept protons, Br?nsted plots were constructed. Br?nsted coefficients were determined for enolization (β = 0.47) and for dehydration (α = 0.54, β= 0.52). While relatively normal catalytic behavior was observed for the enolization of pyruvate, deviations for the dehydration of hydrated pyruvate were noted. Analysis of these deviations, in light of a comparison of the relative magnitude of the catalytic rate coefficients for the reversible hydrations of other carbonyl compounds, suggests the possible contribution of a general-base catalytic path involving the intramolecular participation of the carboxylate group of hydrated pyruvate. The data are also considered in terms of the possible roles the rates of interconversion and positions of equilibria between keto, enol, and hydrated species may play in the physiological reactions of pyruvate. Finally, the Br?nsted analysis provides the necessary basis for a comparison of the relative susceptibilities of the many substrates of carbonic anhydrase II including pyruvate hydrate.
- Damitio,Smith,Meany,Pocker
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p. 3081 - 3087
(2007/10/02)
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