Radical reactions of thiamin pyrophosphate in 2-oxoacid oxidoreductases.
ABSTRACT Thiamin pyrophosphate (TPP) is essential in carbohydrate metabolism in all forms of life. TPP-dependent decarboxylation reactions of 2-oxo-acid substrates result in enamine adducts between the thiazolium moiety of the coenzyme and decarboxylated substrate. These central enamine intermediates experience different fates from protonation in pyruvate decarboxylase to oxidation by the 2-oxoacid dehydrogenase complexes, the pyruvate oxidases, and 2-oxoacid oxidoreductases. Virtually all of the TPP-dependent enzymes, including pyruvate decarboxylase, can be assayed by 1-electron redox reactions linked to ferricyanide. Oxidation of the enamines is thought to occur via a 2-electron process in the 2-oxoacid dehydrogenase complexes, wherein acyl group transfer is associated with reduction of the disulfide of the lipoamide moiety. However, discrete 1-electron steps occur in the oxidoreductases, where one or more [4Fe-4S] clusters mediate the electron transfer reactions to external electron acceptors. These radical intermediates can be detected in the absence of the acyl-group acceptor, coenzyme A (CoASH). The π-electron system of the thiazolium ring stabilizes the radical. The extensively delocalized character of the radical is evidenced by quantitative analysis of nuclear hyperfine splitting tensors as detected by electron paramagnetic resonance (EPR) spectroscopy and by electronic structure calculations. The second electron transfer step is markedly accelerated by the presence of CoASH. While details of the second electron transfer step and its facilitation by CoASH remain elusive, expected redox properties of potential intermediates limit possible scenarios. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.
FEBS Letters 12/1977; 83(2):197-201. · 3.34 Impact Factor
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ABSTRACT: Thermoplasma acidophilum and Sulfolobus acidocaldarius contain coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductases similar to those found in halophilic archaebacteria. A common feature of these enzymes is the formation of a free radical intermediate in the course of the catalytic cycle. The electron-accepting ferredoxins and a similar protein from Desulfurococcus mobilis have been purified and characterized. In contrast to the [2Fe-2S] ferredoxin of Halobacterium halobium, the ferredoxins of thermoacidophilic archaebacteria most likely contain two [4Fe-4S]2 + (2 + .1 +) clusters per molecule. Properties of these proteins are compared with respect to the evolution of archaebacteria.European Journal of Biochemistry 12/1982; 128(1):223-30. · 3.58 Impact Factor
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ABSTRACT: With the publication of the three-dimensional structures of several thiamin diphosphate-dependent enzymes, the chemical mechanism of their non-oxidative and oxidative decarboxylation reactions is better understood. Chemical models for these reactions serve a useful purpose to help evaluate the additional catalytic rate acceleration provided by the protein component. The ability to generate, and spectroscopically observe, the two key zwitterionic intermediates invoked in such reactions allowed progress to be made in elucidating the rates and mechanisms of the elementary steps leading to and from these intermediates. The need remains to develop chemical models, which accurately reflect the enzyme-bound conformation of this coenzyme.FEBS Letters 10/1999; 457(3):298-301. DOI:10.1016/S0014-5793(99)01061-3 · 3.34 Impact Factor