2-Acetylthiamin pyrophosphate (acetyl-TPP) pH-rate profile for hydrolysis of acetyl-TPP and isolation of acetyl-TPP as a transient species in pyruvate dehydrogenase catalyzed reactions

Publisher Website
Google Scholar
Abstract
Rate constants for the hydrolysis of acetyl-TPP were measured between pH values of 2.5 and 7.5 and plotted as log kobs versus pH. The pH-rate profile defined two legs, each with a slope of +1 but separated by a region of decreased slope between pH 4 and pH 6. The rates were insensitive to buffer concentrations. Each leg of the profile reflected specific-base-catalyzed hydrolysis of acetyl-TPP, analogous to the hydrolysis of 2-acetyl-3,4-dimethylthiazolium ion [Lienhard, G. E. (1966) J. Am. Chem. Soc. 88, 5642-5649]. The separation of the two legs of this profile has been shown to be caused by the ionization of a group exhibiting a pKa of 4.73 within acetyl-TPP that is remote from the acetyl group, the aminopyrimidine ring, which is protonated below pH 4.73. The protonation level of this ring has been shown to control the equilibrium partitioning of acetyl-TPP among its carbinolamine, keto, and hydrate forms. The differential partitioning of these species is a major factor causing the separation between the two legs of the pH-rate profile. The characteristic pH-rate profile and the availability of synthetic acetyl-TPP [Gruys, K. J., Halkides, C. J., and Frey, P. A. (1987) Biochemistry 26, 7575-7585] have facilitated the isolation and identification of [1-14C]acetyl-TPP from acid-quenched enzymatic reaction mixtures at steady states. [1-14C]Acetyl-TPP was identified as a transient species in reactions catalyzed by the PDH complex or the pyruvate dehydrogenase component of the complex (E1). The pH-rate profile for hydrolysis of [1-14C]-acetyl-TPP isolated from enzymatic reactions was found to be indistinguishable from that for authentic acetyl-TPP, which constituted positive identification of the 14C-labeled enzymic species.

This publication has 11 references indexed in Scilit: