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

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 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.

The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase

“…In the absence of CoA, the E1 subunit catalyzes ferricyanide and dichloroindophenol reduction (40,41). However, we observed no PCA or pyocyanin reduction in the absence of CoA, suggesting these phenazines are not reduced by the E1 subunit.…”

Determination of Pre-Steady-State Rate Constants on the Escherichia coli Pyruvate Dehydrogenase Complex Reveals That Loop Movement Controls the Rate-Limiting Step

“…While the enamine is oxidized by DCPIP at a rate comparable to its formation (see above), the AcThDP intermediate thus generated is hydrolyzed at a very slow rate, similar to the non-enzymatic hydrolysis rate of synthetic 2-acetylThDP at pH values of 7–7.5 28 . Even when bound to enzyme, the rate of AcThDP hydrolysis is unaffected, hence the low k cat .…”

“…The rate constants for the chemical models are from ref. 32, 28 and 29. NA is not available in literature.…”

Reaction mechanisms of thiamin diphosphate enzymes: redox reactions

“…Frey et al. could isolate AcThDP in the steady state of the overall reaction of PDHc by acid quench trapping [36]. This finding is consistent with a stepwise mechanism of oxidation–reduction and acyl group transfer; however, it cannot disprove a coupled mechanism because AcThDP could be generated from the tetrahedral thiamin‐lipoamide adduct in an equilibrium side reaction.…”