Theoretical Studies of the Electronic Absorption Spectra of Thiamin Diphosphate in Pyruvate Decarboxylase

Abstract

Electronic absorption spectra are oftentimes used to identify reaction intermediates or substrates/products in enzymatic systems, as long as absorption bands can be unequivocally assigned to the species being studied. The latter task is far from trivial given the transient nature of some states and the complexity of the surrounding environment around the active site. To identify unique spectral fingerprints, controlled experiments with model compounds have been used in the past, but even these can sometimes be unreliable. Circular dichroism (CD) and ultraviolet-visible spectra have been tools of choice in the study of the rich chemistry of thiamin diphosphate-dependent enzymes. In this study, we focus on the Zymomonas mobilis pyruvate decarboxylase, and mutant analogues thereof, as a prototypical representative of the thiamin diphosphate (ThDP) enzyme superfamily. Through the use of electronic structure methods, we analyze the nature of electronic excitations in the cofactor. We find that all the determining CD bands around the 280-340 nm spectral range correspond to charge-transfer excitations between the pyrimidine and thiazolium rings of ThDP, which, most likely, is a general property of related ThDP-dependent enzymes. While we can confirm the assignments of previously proposed bands to chemical states, our calculations further suggest that a hitherto unassigned band of enzyme-bound ThDP reports on the ionization state of the canonical glutamate that is required for cofactor activation. This finding expands the spectroscopic "library" of chemical states of ThDP enzymes, permitting a simultaneous assignment of both the cofactor ThDP and the activating glutamate. We anticipate this finding to be helpful for mechanistic analyses of related ThDP enzymes.

Pyruvate dehydrogenase complex deficiency is linked to regulatory loop disorder in the αV138M variant of human pyruvate dehydrogenase

“…As reported previously (12)(13)(14)(15), the binding of ThDP to WT E1 is accompanied by the appearance of two characteristic CD bands in the near-UV spectrum, both of which are shown in Fig. 7: (a) a negative CD band at 330 nm, corresponding to a chargetransfer transition between the thiazolium and 4Ј-aminopyrimidine rings of ThDP, the signature for its 4Ј-aminopyrimidine (AP) tautomer, and (b) a positive CD band at 305 nm assigned to its 1Ј,4Ј-iminopyrimidine (IP) tautomer (12, 16 -18).…”

“…7: (a) a negative CD band at 330 nm, corresponding to a chargetransfer transition between the thiazolium and 4Ј-aminopyrimidine rings of ThDP, the signature for its 4Ј-aminopyrimidine (AP) tautomer, and (b) a positive CD band at 305 nm assigned to its 1Ј,4Ј-iminopyrimidine (IP) tautomer (12, 16 -18). The negative CD band at 320 -330 nm has been observed with several different ThDP-dependent enzymes (15,19). Importantly, both the AP and IP ThDP tautomers could simultaneously be detected in WT E1, suggesting an asymmetry of the two active centers (12).…”

Single‐Turnover Kinetics Reveal a Distinct Mode of Thiamine Diphosphate‐Dependent Catalysis in Vitamin K Biosynthesis

“…As reported previously, the distinct tetrahedral Breslow intermediate in MenD catalysis shows a sharp positive band at λ =302 nm and a broad negative band from λ =305 to 365 nm with double minima at λ =320 and 334 nm by circular dichroism (CD) spectroscopy; this is different from a broad positive band in the ranges of λ =290 to 295 and λ >300 nm that are characteristic of the enamine intermediate in transketolase . These characteristic CD bands are due to the charge‐transfer excitation between the pyrimidine ring and the thiazolium ring of the cofactor, and both were used in our pre‐steady‐state kinetic analysis of the various intermediates in MenD catalysis. The λ =302 nm spike is the same as the λ =300–310 nm positive signal, and this is indicative of a covalent adduct with an sp 3 ‐hydridized C 2α atom and the ThDP cofactor in its iminopyrimidine (IP) form in other ThDP‐dependent enzymes .…”

Structural basis for antibiotic action of the B1 antivitamin 2′-methoxy-thiamine

“…In contrast to genuine ThDP, MThDP did not give rise to the typical near-ultraviolet (UV) circular dichroism (CD) and absorbance charge-transfer bands when bound to EcTK, alluding to a slightly different binding mode (Extended Data Fig. 3b) 19,20 .…”