New evidence for cofactor's amino group function in thiamin catalysis by transketolase.
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.
Meshalkina
Ludmilla E
LE
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Moscow, GSP-2, Russia.
Kochetov
German A
GA
Brauer
Johanna
J
Hübner
Gerhard
G
Tittmann
Kai
K
Golbik
Ralph
R
D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D007527:Isoenzymes / Q000737:chemistry; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013831:Thiamine / Q000737:chemistry*; D014174:Transketolase / Q000737:chemistry*
D007527:Isoenzymes; D029701:Saccharomyces cerevisiae Proteins; D014174:Transketolase; D013831:Thiamine