| dc.description.abstracteng | ThDP-dependent enzymes catalyze a variety of biochemical reactions in all domains of life. The mechanistic studies on these complex systems require a wide range of complementary bioanalytical tools. In this context, electronic absorption spectroscopy has proven itself as a valuable method for the identification of intermediates in ThDP-catalyzed reactions, thereby rationalizing proposed mechanisms. However, the unequivocal assignment of the absorption bands to the correct states of ThDP and intermediates is required for appropriate conclusions. This task is far from trivial
considering the transient character of some intermediates as well as the complexity of the surrounding enzyme environment. Controlled experiments on model compounds, frequently employed for the assignments, can be unreliable because of the absence of critical conditions in enzymes. For this purpose, theoretical methods have been applied in this thesis to obtain a detailed view of the involved excitations including the analyses of the determining factors behind the different spectra. These studies provide novel insights into the spectral signatures of ThDP-dependent enzymes and an important contribution to their mechanistic understanding.
In a first step, the spectral signatures of the pre-equilibrium states of ThDP are investigated through model compound calculations up to a full QM/MM description of the cofactors in the ZmPDC enzyme. The observed CD bands in the near UV region could all be associated with charge-transfer excitations between the pyrimidine and the thiazolium rings of ThDP. In particular, the fundamental role of the protonation state of the canonical glutamate is highlighted for the location of the ThDP bands. These studies result in the simultaneous assignment of the chemical states of both the cofactor and the activating glutamate to the two previously proposed spectral fingerprints of ThDP, but also to a third hitherto unassigned band.
Investigations have also been carried out on spectra of enzymatic on-pathway intermediates. Calculations for the tautomers of 2-acetyl-ThDP have helped to unravel the experimental observations in the B. breve PK enzyme. Both the keto and enolate states of this intermediate are assigned to a common band in the UV-vis spectra. The latter plays a central role for the mechanism of PK through kinetic stabilization in absence of the co-substrate, preventing the system from off-pathway hydrolysis. The second intermediate of particular interest has been 1,2-dihydroxyethyl-ThDP, which was previously identified with a key role in the TK enzyme mechanism. UV-vis measurements had revealed an uncommon broad absorption band with a range of about 1 eV. Spectra calculations in the active site of E. coli TK have helped to identify the source of this observation. A proton transfer equilibrium from the intermediate to a histidine residue is proposed which connects the two limits of the observed band.
The new information gathered from the studies of ThDP and the aforementioned intermediates is a key contribution to understanding their reactivity. The low-lying excited states of the proposed intermediates have been analyzed with respect to their excitation characters. This has lead to a classification of the spectral fingerprints. Subsequent analyses have identified a structure-excitation energy relationship for ThDP intermediates. These results provide a roadmap for the interpretation of absorption spectra of ThDP-dependent enzymes. | de |