Stereoisomers of tetrahydrothiamin pyrophosphate, potent inhibitors of the pyruvate dehydrogenase multienzyme complex from Escherichia coli

Abstract

Tetrahydrothiamin pyrophosphate, an analogue of thiamin pyrophosphate (TPP) in which the thiazolium ring has been reduced to a thiazolidine ring, was prepared by borohydride reduction of TPP. It consists of four stereoisomers, comprising two diastereomers each of which is a racemic mixture, generated by the introduction of two chiral centers on the thiazolidine ring. The major and minor diastereomers were separated and inferred to be of the cis and trans configurations, respectively, from a study of the nuclear Overhauser effects in the 1H NMR spectrum of the simpler tetrahydrothiamin. Tetrahydro-TPP behaves as a mixture of potent inhibitors of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase complex from Escherichia coli. The site of binding is probably the TPP-binding site on E1, and the Kd for each of the four stereoisomers was estimated. The cis isomers of tetrahydro-TPP bind more tightly than does TPP, whereas the trans isomers appear to bind with about the same Kd as TPP. Sodium borohydride caused a rapid inhibition of E1 activity in the presence of TPP, believed to be due to reaction of borohydride with enzyme-bound TPP. The experiments are consistent with an enhancement of the reactivity of the thiazole ring of TPP when bound to the catalytic site of E1, which could be due to polarization of the greater than +N=C bond near a hydrophobic or positively charged region of the protein. A spontaneous reactivation occurred after the initial inhibition by borohydride, attributable to a weakly binding inhibitor, not tetrahydro-TPP, being formed at the catalytic site.

Purification and characterization of acetoin:2,6-dichlorophenolindophenol oxidoreductase, dihydrolipoamide dehydrogenase, and dihydrolipoamide acetyltransferase of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system

“…The second product formed by the Ao:DCPIP-catalyzed cleavage reactions in the presence of the artificial electron acceptor DCPIP in vitro is acetate, which is probably hydrolytically split from acetyl-TPP after DCPIP-caused oxidation of hydroxyethyl-TPP (18). The in vitro formation of acetate in the presence of an artificial electron acceptor (i.e., ferricyanide or DCPIP) is well described for many different TPP-dependent enzymes, such as the E1 components of 2-oxo acid dehydrogenase complexes (18,24,32,43) and pyruvate decarboxylase (8) or glycolate with transketolase (27). Since no pyruvate-or 2-oxoglutarate-dependent reduction of DCPIP was measured with crude extracts and with purified Ao:DCPIP OR, it is unlikely that the cleavage of acetoin is catalyzed by nonspecific E1 components of 2-oxo acid dehydrogenase complexes.…”

Molecular characterization of the mde operon involved in L-methionine catabolism of Pseudomonas putida

“…E1 activity was measured by an assay involving the reduction of 2,6-dichlorophenolindophenol (DCPIP) (21). In the assay, 50 l of 100 mM ␣-keto acid (pyruvate or ␣-ketobutyrate) was added to 1,050 l of mixture containing 0.1 M potassium phosphate (pH 7.4), 0.1 mM DCPIP, 0.2 mM TPP, 0.1 mM MgCl 2 , and 0.5 to 1.0 mg of cell extract at 30°C.…”

Effect of Substitutions in the Thiamin Diphosphate-Magnesium Fold on the Activation of the Pyruvate Dehydrogenase Complex from Escherichia coli by Cofactors and Substrate

“…analogues including bromopyruvate (13), fluoropyruvate (14,15), phosphonate analogues of pyruvate (16,17), mono-and bifunctional arsenoxides (18,19,20), branched chain keto acids (21), and tetrahydrothiamin pyrophoshate (22).…”