Aspartic acid 214 in Citrobacter freundii tyrosine phenol-lyase ensures sufficient C-H-acidity of the external aldimine intermediate and proper orientation of the cofactor at the active site

The substitutions result in a decrease in the cofactor affinity of about four orders of magnitude. D214A and D214N TPLs do not catalyze the decomposition of l-Tyr and 3-fluoro-l-Tyr. They decompose substrates, containing better leaving groups with rates reduced by one or two orders of magnitude. Lognormal resolution of the spectra of the mutant enzymes revealed that the N1 atom of the cofactor is deprotonated. Spectral characteristics of internal and external aldimines of the mutant TPLs and the data on their interaction with quasisubstrates demonstrate that replacements of Asp214 lead to alteration of active site conformations. The mutant enzymes do not form noticeable amounts of a quinonoid upon interaction with inhibitors, but catalyze isotope exchange of C-α-proton of a number of amino acids for deuterium in 2H2O. The kex values for the isotope exchange of l-phenylalanine and 3-fluoro-l-tyrosine are close to the kcat values for reacting substrates. Thus, for the mutant TPLs the stage of C-α-proton abstraction may be considered as a rate-limiting for the whole reaction.