The origin of the absorption band induced through the interaction between apotransketolase and thiamin diphosphate.
It has long been known that formation of a catalytically active holotransketolase from the apoenzyme and coenzyme (thiamin diphosphate) is accompanied by the appearance of a new band, in both the absorption and CD spectra. Binding and subsequent conversion of the substrates bring about changes in this band's intensity. The observation of these changes allows the investigator to monitor the coenzyme-to-apoenzyme binding and the conversion of substrates during the transketolase reaction and thus to kinetically characterize its individual steps. The origin of the thiamin diphosphate induced absorption band has been postulated to be resulted from formation of a charge transfer complex or alternatively from an induced conformational transition of the enzyme. The latter brings aromatic amino acid residues into close proximity and generates the absorption. However, X-ray crystallographic and enzyme point mutation experiments cast doubts on both of these hypotheses. Here we show that the binding of thiamin diphosphate to the apotransketolase leads to the conversion of the 4'-amino tautomeric form of its aminopyrimidine ring into the N(1')H-imino tautomeric form. This imino form emerges as a result of the coenzyme's aminopyrymidine ring incorporation into the hydrophobic pocket of the transketolase active center and is stabilized through the interactions with Glu418 and Phe445 residues. The N(1')H-imino tautomeric form of thiamin diphosphate is thought to be the origin of the holotransketolase absorption band induced through the coenzyme binding.
Kovina
Marina V
MV
A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Moscow, GSP-2, Russia.
Bykova
Irina A
IA
Solovjeva
Olga N
ON
Meshalkina
Ludmilla E
LE
Kochetov
German A
GA
D002384:Catalysis; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013054:Spectrophotometry, Atomic; D013835:Thiamine Pyrophosphate / Q000378:metabolism*; D014174:Transketolase / Q000378:metabolism*
D014174:Transketolase; D013835:Thiamine Pyrophosphate