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

摘要: In the X-ray structure of tyrosine phenol-lyase (TPL) Asp214 is located at H-bonding distance from N1 atom cofactor. This residue has been replaced with Ala and Asn properties mutant enzymes have studied. The substitutions result in a decrease cofactor affinity about four orders magnitude. D214A D214N TPLs do not catalyze decomposition l-Tyr 3-fluoro-l-Tyr. They decompose substrates, containing better leaving groups rates reduced by one or two Lognormal resolution spectra revealed that deprotonated. Spectral characteristics internal external aldimines data on their interaction quasisubstrates demonstrate replacements lead to alteration active site conformations. form noticeable amounts quinonoid upon inhibitors, but isotope exchange C-alpha-proton number amino acids for deuterium (2)H(2)O. k(ex) values l-phenylalanine 3-fluoro-l-tyrosine are close k(cat) reacting substrates. Thus, stage abstraction may be considered as rate-limiting whole reaction.