TAILIEUCHUNG - Báo cáo khoa học: Effect of coenzyme modification on the structural and catalytic properties of wild-type transketolase and of the variant E418A from Saccharomyces cerevisiae Contrasting protonation state requirements of thiamin diphosphate in decarboxylases and transketolases

Transketolase from baker’s yeast is a thiamin diphosphate-dependent enzyme in sugar metabolism that reconstitutes with various analogues of the coenzyme. The methylated analogues (4¢-methylamino-thiamin diphos-phate and N1¢-methylated thiamin diphosphate) of the native cofactor were used to investigate the function of the aminopyrimidine moiety of the coen-zyme in transketolase catalysis. | iFEBS Journal Effect of coenzyme modification on the structural and catalytic properties of wild-type transketolase and of the variant E418A from Saccharomyces cerevisiae Contrasting protonation state requirements of thiamin diphosphate in decarboxylases and transketolases Ralph Golbik1 Ludmilla E. Meshalkina2 Tatjana Sandalova3 Kai Tittmann1 Erik Fiedler1 Holger Neef1 Stephan Konig1 Ronald Kluger4 German A. Kochetov2 Gunter Schneider3 and Gerhard Hubner1 1 Martin-Luther-University Halle-Wittenberg Institute of Biochemistry Department of Enzymology Halle Saale Germany 2 . Belozersky Institute of Physico-ChemicalBiology Moscow State University Russia 3 Division of Molecular StructuralBiology Department of MedicalBiochemistry and Biophysics Karolinska Institute Stockholm Sweden 4 Department of Chemistry University of Toronto Toronto Ontario Canada Keywords intermediates stopped-flow kinetics thiamin diphosphate analogues transketolase X-ray crystallography Correspondence R. Golbik Institute of Biochemistry Martin-Luther-University Halle-Wittenberg Kurt-Mothes-Strasse 3 Halle Saale 06120 Germany Fax 49 345 55 27011 Tel 49 345 55 24829 E-mail golbik@ Note These authors contributed equally to this work Received 22 October 2004 revised 4 January 2005 accepted 10 January 2005 doi Transketolase from baker s yeast is a thiamin diphosphate-dependent enzyme in sugar metabolism that reconstitutes with various analogues of the coenzyme. The methylated analogues 4 -methylamino-thiamin diphosphate and N1 -methylated thiamin diphosphate of the native cofactor were used to investigate the function of the aminopyrimidine moiety of the coenzyme in transketolase catalysis. For the wild-type transketolase complex with the 4 -methylamino analogue no electron density was found for the methyl group in the X-ray structure whereas in the complex with the N1 -methylated coenzyme the entire aminopyrimidine ring was disordered. This .

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