Induced Absorption Band of Holotransketolase and Its Interpretation

M. V. Kovina, I. A. Bykova, L. E. Meshalkina, and G. A. Kochetov^*

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992 Russia; fax: (095) 939-3181; E-mail: kochetov@genebee.msu.su

^* To whom correspondence should be addressed.

Received March 19, 2002

---

It has long been known that formation of a catalytically active holotransketolase from the apoenzyme and thiamine diphosphate (ThDP) is accompanied by appearance, in both the absorption and CD spectra, of a new band. Binding and subsequent conversion of transketolase substrates bring about changes in the intensity of this band. The observation of these changes allows the investigator to monitor the coenzyme-to-apoenzyme binding and the conversion of the substrates during the transketolase reaction and thus to kinetically characterize its individual steps. As regards the new absorption band induced by ThDP binding, its nature, until recently, remained unknown. The reason for its appearance was considered to be either the formation of a charge transfer complex between ThDP and tryptophan (phenylalanine) residue or stacking interaction between the residues of aromatic amino acids. They are thought to be brought together as a result of conformational changes of the apoenzyme during its interaction with the coenzyme. However none of these hypotheses had been substantiated experimentally. According to our hypothesis, the induced absorption band is that of the imino form of ThDP resulting from three contributing features of the ThDP binding site of transketolase: the relative hydrophobicity of this site, hydrogen bonding of the N1´-atom of the ThDP aminopyrimidine ring to Glu418, and base stacking interactions between the aminopyrimidine ring of ThDP and Phe445.

---

KEY WORDS: thiamine diphosphate, transketolase, new absorption band, circular dichroism, absorption spectrum

---