The reversible enolization and hydration of pyruvate: possible roles of keto, enol, and hydrated pyruvate in lactate dehydrogenase catalysis

The reversible enolization and hydration of pyruvic acid and pyruvate anion were monitored using spectrophotometric methods at several temperatures. W idely varying values for the equilibrium constant for the enolization of py ruvic acid and pyruvate ion appear in the literature. To accurately determi ne the position of equilibrium for the enolization reaction, we have develo ped a method that gives consistent results in which purified samples of sod ium pyruvate are first "titrated" with triiodide ion to remove any triiodid e-scavenging impurities such as those resulting from aldol condensation rea ctions. After reequilibration to allow the regeneration of enol pyruvate, t he 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-ord er decrease due to the formation of new enol pyvuvate molecules. The absorb ance change during the burst phase of the reaction is proportional to the e nol concentration plus that of any triiodide-scavenging impurity which may be present in the original pyruvate solution. Thus, as the quantity of trii odide used in the pretreatment stage of the experiments is increased, these burst absorbance changes, Delta A, decrease until a constant value of Delt a A is reached. Accordingly, this final Delta A value is proportional to en ol pyruvate (or enol pyruvic acid) in the absence of triiodide-scavenging i mpurity, allowing the accurate and reproducible determinations of K-enol. T he equilibrium constants for both pyruvate and pyruvic acid are relatively temperature insensitive and, typically, K-enol (pyruvate anion) = 2.6 x 10( -5) and K-enol (pyruvic acid) = 7.8 x 10(-5) at 25.0 degrees C. The zero-or der phase of the reaction of triiodide ion may be used to calculate rate co nstants for enolization. The hydration and dehydration of pyruvic acid were followed directly by following absorbance changes in the peak at 340 nm du e to the keto group. The thermodynamic and kinetic results reported in this paper are used to help determine whether the observed "substrate" inhibiti on of the lactate dehydrogenase catalyzed reduction of pyruvate is actually caused by keto, hydrated, or enol pyruvate.