title: Effect of coenzyme modification on the structural and catalytic properties of wild-type transketolase and of the variant E418A from Saccharomyces cerevisiae.
abstract: 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 indicates a high flexibility of the respective parts of the enzyme-bound thiamin diphosphate analogues. In the E418A variant of transketolase reconstituted with N1'-methylated thiamin diphosphate, the electron density of the analogue was well defined and showed the typical V-conformation found in the wild-type holoenzyme [Lindqvist Y, Schneider G, Ermler U, Sundstrom M (1992) EMBO J11, 2373-2379]. The near-UV CD spectrum of the variant E418A reconstituted with N1'-methylated thiamin diphosphate was identical to that of the wild-type holoenzyme, while the CD spectrum of the variant combined with the unmodified cofactor did not overlap with that of the native protein. The activation of the analogues was measured by the H/D-exchange at C2. Methylation at the N1' position of the cofactor activated the enzyme-bound cofactor analogue (as shown by a fast H/D-exchange rate constant). The absorbance changes in the course of substrate turnover of the different complexes investigated (transient kinetics) revealed the stability of the alpha-carbanion/enamine as the key intermediate in cofactor action to be dependent on the functionality of the 4-aminopyrimidine moiety of thiamin diphosphate.
authors.affiliation: Department of Enzymology, Martin-Luther-University Halle-Wittenberg, Institute of Biochemistry, Halle/Saale, Germany. golbik@biochemtech.uni-halle.de
authors.forename: Ralph
authors.initials: R
authors.lastname: Golbik
authors.forename: Ludmilla E
authors.initials: LE
authors.lastname: Meshalkina
authors.forename: Tatjana
authors.initials: T
authors.lastname: Sandalova
authors.forename: Kai
authors.initials: K
authors.lastname: Tittmann
authors.forename: Erik
authors.initials: E
authors.lastname: Fiedler
authors.forename: Holger
authors.initials: H
authors.lastname: Neef
authors.forename: Stephan
authors.initials: S
authors.lastname: König
authors.forename: Ronald
authors.initials: R
authors.lastname: Kluger
authors.forename: German A
authors.initials: GA
authors.lastname: Kochetov
authors.forename: Gunter
authors.initials: G
authors.lastname: Schneider
authors.forename: Gerhard
authors.initials: G
authors.lastname: Hübner
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D018360:Crystallography, X-Ray; D007700:Kinetics; D020125:Mutation, Missense; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D014174:Transketolase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D014174:Transketolase