Identification of enzyme-bound intermediates via their spectroscopic signatures, which then allows direct monitoring of the kinetic fate of these intermediates, poses a continuing challenge. As an electrophilic covalent catalyst, the thiamin diphosphate (ThDP) coenzyme forms a number of noncovalent and covalent intermediates along its reaction pathways, and multiple UV-vis and circular dichroism (CD) bands have been identified at Rutgers pertinent to several among them. These electronic transitions fall into two classes: those for which the conjugated system provides a reasonable guide to the observed λmax and others in which there is no corresponding conjugated system and the observed CD bands are best ascribed to charge transfer (CT) transitions. Herein is reported the reaction of four ThDP enzymes with alternate substrates: (a) acetyl pyruvate, its methyl ester, and fluoropyruvate, these providing the shortest side chains attached at the thiazolium C2 atom and leading to CT bands with λmax values of >390 nm, not pertinent to any on-pathway conjugated systems (estimated λmax values of <330 nm), and (b) (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid displaying both a conjugated enamine (430 nm) and a CT transition (480 nm). We suggest that the CT transitions result from an interaction of the π bond on the ThDP C2 side chain as a donor, and the positively charged thiazolium ring as an acceptor, and correspond to covalent ThDP-bound intermediates. Time resolution of these bands allows the rate constants for individual steps to be determined. These CD methods can be applied to the entire ThDP superfamily of enzymes and should find applications with other enzymes.