New evidence for cofactor's amino group function in thiamin catalysis by transketolase.

New evidence for cofactor's amino group function in thiamin catalysis by transketolase. Research Abstract Details 

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New evidence for cofactor's amino group function in thiamin catalysis by transketolase. Abstract Text:

Ludmilla E Meshalkina,German A Kochetov,Johanna Brauer,Gerhard ,Kai Tittmann,Ralph Golbik,Ludmilla E Meshalkina,German A Kochetov,Johanna Brauer,Gerhard Hübner,Kai Tittmann,Ralph Golbik,

Transketolase from Saccharomyces cerevisiae exhibits a rarely reported activity with a methylated analogue of the native cofactor, 4'-methylamino-thiamin diphosphate. We demonstrated the kinetic stability of the dihydroxyethyl carbanion/enamine intermediate to be dependent on the functionality of the 4'-aminopyrimidine moiety of thiamin diphosphate [R. Golbik, L.E. Meshalkina, T. Sandalova, K. Tittmann, E. Fiedler, H. Neef, S. König, R. Kluger, G.A. Kochetov, G. Schneider, G. Hübner, Effect of coenzyme modification on the structural and catalytic properties of wild-type transketolase and of the variant E418A from Saccharomyces cerevisae, FEBS J. (2005) 272 1326-1342]. This paper extends these investigations of the function of the coenzyme's aminopyrimidine in transketolase catalysis exemplified for the 4'-monomethylamino-thiamin diphosphate analogue. Here, we report near UV circular dichroism data and NMR-based analysis of reaction intermediates that give evidence for a strong destabilisation of the carbanion/enamine of DHE-4'-monomethylamino-thiamin diphosphate on the enzyme. A new negative band in near UV circular dichroism arising during turnover is attributed to the conjugate acid of the carbanion/enamine intermediate, an assignment additionally corroborated by (1)H NMR-based intermediate analysis. As opposed to the kinetically stabilized carbanion/enamine intermediate in transketolase when reconstituted with the native cofactor, DHE-4'-monomethylamino-thiamin diphosphate is rapidly released from the active centers during turnover and accumulates in the medium on a preparative scale.

New evidence for cofactor's amino group function in thiamin catalysis by transketolase. Publishing Authors By Initials

LE Meshalkina,GA Kochetov,J Brauer,G ,K Tittmann,R Golbik,LE Meshalkina,GA Kochetov,J Brauer,G Hübner,K Tittmann,R Golbik,

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New evidence for cofactor's amino group function in thiamin catalysis by transketolase. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Biochemical and biophysical research communication

VOLUME: 366

Page Numbers: 692-7

Journal Abbreviation: Biochem. Biophys. Res. Commun.

ISSN: 1090-2104

DAY: 10

MONTH: 12

YEAR: 2007

New evidence for cofactor's amino group function in thiamin catalysis by transketolase. Information

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LANGUAGE: eng

NlmUniqueID: 372516

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Grant and Affiliation Information for New evidence for cofactor's amino group function in thiamin catalysis by transketolase.

AFFILIATION: A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Moscow, GSP-2, Russia.

Country: United States

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MEDLINETA: Biochem Biophys Res Commun

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