title: 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.
abstract: In the X-ray structure of tyrosine phenol-lyase (TPL) Asp214 is located at H-bonding distance from the N1 atom of the cofactor. This residue has been replaced with Ala and Asn and the properties of the mutant enzymes have been studied. 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-alpha-proton of a number of amino acids for deuterium in (2)H(2)O. The k(ex) values for the isotope exchange of l-phenylalanine and 3-fluoro-l-tyrosine are close to the k(cat) values for reacting substrates. Thus, for the mutant TPLs the stage of C-alpha-proton abstraction may be considered as a rate-limiting for the whole reaction.
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia. tvd@eimb.ru
authors.forename: T V
authors.initials: TV
authors.lastname: Demidkina
authors.forename: N G
authors.initials: NG
authors.lastname: Faleev
authors.forename: A I
authors.initials: AI
authors.lastname: Papisova
authors.forename: N P
authors.initials: NP
authors.lastname: Bazhulina
authors.forename: V V
authors.initials: VV
authors.lastname: Kulikova
authors.forename: P D
authors.initials: PD
authors.lastname: Gollnick
authors.forename: R S
authors.initials: RS
authors.lastname: Phillips
mesh_terms: D000409:Alanine / Q000235:genetics; D001051:Apoenzymes / Q000737:chemistry; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002942:Circular Dichroism; D016971:Citrobacter freundii / Q000201:enzymology* / Q000235:genetics; D003067:Coenzymes / Q000737:chemistry*; D017666:Deuterium Oxide / Q000737:chemistry; D006714:Homoserine / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013053:Spectrophotometry; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics; D014447:Tyrosine Phenol-Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001051:Apoenzymes; D003067:Coenzymes; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D014443:Tyrosine; D010649:Phenylalanine; D006714:Homoserine; D001216:Asparagine; D014447:Tyrosine Phenol-Lyase; D017666:Deuterium Oxide; D000409:Alanine