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.

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 amino-pyrimidine 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., & 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.