The molecular origin of the thiamin diphosphate‐induced spectral bands of ThDP‐dependent enzymes

Abstract

New and previously published data on a variety of ThDP-dependent enzymes such as baker's yeast transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase from pigeon breast muscle, bovine heart, bovine kidney, Neisseria meningitidis and E. coli show their spectral sensitivity to ThDP binding. Although ThDP-induced spectral changes are different for different enzymes, their universal origin is suggested as being caused by the intrinsic absorption of the pyrimidine ring of ThDP, bound in different tautomeric forms with different enzymes. Non-enzymatic models with pyrimidine-like compounds indicate that the specific protein environment of the aminopyrimidine ring of ThDP determines its tautomeric form and therefore the changeable features of the inducible effect. A polar environment causes the prevalence of the aminopyrimidine tautomeric form (short wavelength region is affected). For stabilization of the iminopyrimidine tautomeric form (both short- and long-wavelength regions are affected) two factors appear essential: (i) a nonpolar environment and (ii) a conservative carboxyl group of a specific glutamate residue interacting with the N1' atom of the aminopyrimidine ring. The two types of optical effect depend in a different way upon the pH, in full accordance with the hypothesis tested. From these studies it is concluded that the inducible optical rotation results from interaction of the aminopyrimidine ring with its asymmetric environment and is defined by the protonation state of N1' and the 4'-nitrogen.

Structural basis for the magnesium-dependent activation of transketolase from Chlamydomonas reinhardtii

“…This agrees with previous studies that analyzed the rate of transition from the apo-form of yeast TK to the fully active holo-form, demonstrating that the long time needed to reach the maximal activity is strongly decreased upon pre-incubation of the protein with both TPP and Mg 2+ [8,36]. The formation of fully reconstituted TPP-dependent enzymes can be monitored by the appearance of UV and ICD signals upon TPP binding [39]. ICD spectra stem from the perturbations to the symmetry and electronic structure of a non-chiral ligand (i.e.…”

“…Upon binding of TPP to TK from S. cerevisiae, an increase in absorption can be observed in the near UV range (285-360 nm) [1,[37][38][39]. A peculiar bisignate induced CD (ICD) signal, closely related to the increase in UV absorption, is also observed across a variety of TPP-dependent enzymes [1,39,40].…”

Glucopyranosylidene-spiro-imidazolinones, a New Ring System: Synthesis and Evaluation as Glycogen Phosphorylase Inhibitors by Enzyme Kinetics and X-ray Crystallography

“…Provided that different tautomeric forms may also have considerable impact on the ECD, the ECD computational studies, complemented by DFT-NMR calculations, , also aimed at identifying the major tautomers present in the solution. Application of ECD for the identification of ring tautomers is rather rare in the literature. − …”

Binding of the Coenzyme and Formation of the Transketolase Active Center

“…4b). In the ThDP-dependent enzymes, whose spectra in the resting state are different from holoTK spectra, ThDP exists in a transient imino tautomeric state probably during catalysis only (42,43). This point of view is proved in literature (46,47).…”

“…6). The imino form is stabilized by the interaction of the coenzyme with phenylalanine 445 and N1' of the aminopyrimidine ring with the carboxyl group of Glu418 (42,43) (Fig. 6).…”