Kinetic control of thiamin diphosphate activation in enzymes studied by proton-nitrogen correlated NMR spectroscopy.

Proton-nitrogen correlated NMR studies were performed on thiamin diphosphate, which has been specifically labeled with (15)N at the 4'-amino group. After reconstitution of the labeled coenzyme with the apoenzymes of both wild-type pyruvate decarboxylase from Zymomonas mobilis and the E50Q variant, a high-field shift of the (15)N signal of approximately 4 ppm is observed at pH 5.9 when compared to that of the free coenzyme, indicating a higher electron density at the 4'-amino nitrogen in the enzyme-bound state. The pH dependence of the chemical shift of the (15)N signals in the (1)H-(15)N heteronuclear single-quantum coherence NMR spectra reveals typical titration curves for the free as well as the reconstituted coenzyme with nearly identical chemical shift end points. The midpoints of the transitions are at pH 5.3 and 5.0 for the free and enzyme-bound coenzyme, respectively. We conclude that the tremendous rate acceleration of C2-H deprotonation in ThDP enzymes is mainly the result of the enforced V conformation of the cofactor in the active site being perfectly suited to allowing intramolecular acid-base catalysis.

Effect of coenzyme modification on the structural and catalytic properties of wild-type transketolase and of the variant E418A from Saccharomyces cerevisiae.

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.