Reference: Meshalkina LE, et al. (2008) New evidence for cofactor's amino group function in thiamin catalysis by transketolase. Biochem Biophys Res Commun 366(3):692-7

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Abstract


Transketolase from Saccharomyces cerevisiae exhibits a rarely reported activity with a methylated analogue of the native cofactor, 4'-nor-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 thiamine diphosphate [R. Golbik, L.E. Meshalkina, T. Sandalova, K. Tittmann, E. Fiedler, H. Neef, S. Konig, R. Kluger, G.A. Kochetov, G. Schneider, G. Hubner, 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'-nor-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-thiamine diphosphate on the enzyme. A new negative band in near UV circular dichroism arising during turnover is attributed to pertain 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-thiamine diphosphate is rapidly released from the active centers during turnover and accumulates in the medium on a preparative scale.

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Journal Article
Authors
Meshalkina LE, Kochetov GA, Brauer J, Hubner G, Tittmann K, Golbik R
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