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Isotope effect, mechanism, and origin of catalysis in the decarboxylation of mandelylthiamin.

ABSTRACT: The mechanism of decarboxylations in water and the catalysis of mandelylthiamin (MTh) decarboxylation by pyridinium ions is explored. It has been recently proposed that a decarboxylation step forming an intermediate molecule/CO(2) pair is reversible and that pyridinium ions catalyze the MTh decarboxylation by trapping the intermediate, preventing reversion to MTh. A calculation of the barrier for the back reaction goes against this proposal, as the diffusional separation of CO(2) would be on the order of 10,000 times faster. A comparison of predicted and experimental isotope effects for a series of decarboxylations including the MTh reaction shows in each case the absence of significant reversibility of the decarboxylation step. An alternative origin of the pyridinium catalysis of decarboxylation is proposed, based on the formal binding of the pyridinium ions to the decarboxylation transition state.

SUBMITTER: Gonzalez-James OM

PROVIDER: S-EPMC2880891 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Isotope effect, mechanism, and origin of catalysis in the decarboxylation of mandelylthiamin.

Gonzalez-James Ollie M OM Singleton Daniel A DA

Journal of the American Chemical Society 20100501 20

The mechanism of decarboxylations in water and the catalysis of mandelylthiamin (MTh) decarboxylation by pyridinium ions is explored. It has been recently proposed that a decarboxylation step forming an intermediate molecule/CO(2) pair is reversible and that pyridinium ions catalyze the MTh decarboxylation by trapping the intermediate, preventing reversion to MTh. A calculation of the barrier for the back reaction goes against this proposal, as the diffusional separation of CO(2) would be on the ...[more]

PMID: 20433168

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Project description:1. Michaelis-Menten parameters for the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside and 3,4-dinitrophenyl beta-D-galactopyranoside Escherichia coli (lacZ) beta-galactosidase were measured as a function of pH or pD (pL) in both 1H2O and 2H2O. 2. For hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-free enzyme, V is pL-independent below pL 9, but the V/Km-pL profile is sigmoid, the pK values shifting from 7.6 +/- 0.1 in 1H2O to 8.2 +/- 0.1 in 2H2O, and solvent kinetic isotope effects are negligible, in accord with the proposal [Sinnott, Withers & Viratelle (1978) Biochem. J. 175, 539-546] that glycone-aglycone fission without acid catalysis governs both V and V/Km. 3. V for hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme varies sigmoidally with pL, the pK value shifting from 9.19 +/- 0.09 to 9.70 +/- 0.07; V/Km shows both a low-pL fall, probably due to competition between Mg2+ and protons [Tenu, Viratelle, Garnier & Yon (1971) Eur. J. Biochem. 20, 363-370], and a high-pL fall, governed by a pK that shifts from 8.33 +/- 0.08 to 8.83 +/- 0.08. There is a negligible solvent kinetic isotope effect on V/Km, but one of 1.7 on V, which a linear proton inventory shows to arise from one transferred proton. 4. The variation of V and V/Km with pL is sigmoid for hydrolysis of 3,4-dinitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme, with pK values showing small shifts, from 8.78 +/- 0.09 to 8.65 +/- 0.08 and from 8.7 +/- 0.1 to 8.9 +/- 0.1 respectively. There is no solvent isotope effect on V or V/Km for 3,4-dinitrophenyl beta-D-galactopyranoside, despite hydrolysis of the galactosyl-enzyme intermediate governing V. 5. Identification of the 'conformation change' in the hydrolysis of aryl galactosides proposed by Sinnott & Souchard [(1973) Biochem. J. 133, 89-98] with the protolysis of the magnesium phenoxide arising from the action of enzyme-bound Mg2+ as an electrophilic catalyst rationalizes these data and also resolves the conflict between the proposals and the 18O kinetic-isotope-effect data reported by Rosenberg & Kirsch [(1981) Biochemistry 20, 3189-3196]. It should be noted that the actual Km values were determined to higher precision than can be estimated from the Figures in this paper.(ABSTRACT TRUNCATED AT 400 WORDS)

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Isotope effect, mechanism, and origin of catalysis in the decarboxylation of mandelylthiamin.

Publications

Isotope effect, mechanism, and origin of catalysis in the decarboxylation of mandelylthiamin.

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