Kinetic study of the H103A mutant yeast transketolase.
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, alpha,beta-dihydroxyethyl-thiamin diphosphate, by forming a hydrogen bond with the oxygen of its beta-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.
Selivanov
Vitaliy A
VA
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Moscow, GSP-2, Russia.
Kovina
Marina V
MV
Kochevova
Natalia V
NV
Meshalkina
Ludmilla E
LE
Kochetov
German A
GA
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D006639:Histidine / Q000235:genetics; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000378:metabolism; D014174:Transketolase / Q000235:genetics* / Q000378:metabolism*
D020035:Holoenzymes; D011994:Recombinant Proteins; D006639:Histidine; D014174:Transketolase; D008274:Magnesium; D000409:Alanine; D013835:Thiamine Pyrophosphate; D002118:Calcium