Kinetic study of the H103A mutant yeast transketolase

• Published in: FEBS letters Vol. 567; no. 2; pp. 270 - 274
• Main Authors: Selivanov, Vitaliy A., Kovina, Marina V., Kochevova, Natalia V., Meshalkina, Ludmilla E., Kochetov, German A.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 04.06.2004
• Subjects: Alanine - genetics; Amino Acid Substitution; Binding Sites; Calcium - chemistry; Calcium - metabolism; DHEThDP, α,β-dihydroxyethyl-thiamin diphosphate; Dimerization; GAPDH, d-glyceraldehyde 3-phosphate dehydrogenase; Histidine - genetics; Holoenzymes - chemistry; Holoenzymes - metabolism; Induced optical activity; Kinetic constant; Kinetic model; Kinetics; Magnesium - chemistry; Magnesium - metabolism; Mutagenesis; Mutagenesis, Site-Directed; Protein Binding; R5P, d-ribose 5-phosphate; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Substrate Specificity; ThDP, thiamin diphosphate; Thiamin diphosphate; Thiamine Pyrophosphate - metabolism; TK, transketolase; Transketolase; Transketolase - genetics; Transketolase - metabolism; X5P, d-xylulose 5-phosphate; Thiamin diphosphate; Kinetic model; TK, transketolase; Induced optical activity; Mutagenesis; Transketolase; GAPDH, d-glyceraldehyde 3-phosphate dehydrogenase; R5P, d-ribose 5-phosphate; ThDP, thiamin diphosphate; Kinetic constant; DHEThDP, α,β-dihydroxyethyl-thiamin diphosphate; X5P, d-xylulose 5-phosphate
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2004.04.082

Data from site-directed mutagenesis and X-ray crystallography show that His103 of holotransketolase (holoTK) does not come into contact with thiamin diphosphate (ThDP) but stabilizes the transketolase (TK) reaction intermediate, α,β-dihydroxyethyl-thiamin diphosphate, by forming a hydrogen bond with the oxygen of its β-hydroxyethyl group [Eur. J. Biochem. 233 (1995) 750; Proc. Natl. Acad. Sci. USA 99 (2002) 591]. We studied the influence of His103 mutation on ThDP-binding and enzymatic activity. It was found that mutation does not affect the affinity of the coenzyme to apotransketolase (apoTK) in the presence of Ca 2+ (a cation found in the native holoenzyme) but changes all the kinetic parameters of the ThDP–apoTK interaction in the presence of Mg 2+ (a cation commonly used in ThDP-dependent enzymes studies). It was concluded that the structures of TK active centers formed in the presence of Mg 2+ and Ca 2+ are not identical. Mutation of His103 led to a significant acceleration of the one-substrate reaction but a slow down of the two-substrate reaction so that the rates of both types of catalysis became equal. Our results provide evidence for the intermediate-stabilizing function of His103.