title: Citrobacter freundii tyrosine phenol-lyase: the role of asparagine 185 in modulating enzyme function through stabilization of a quinonoid intermediate.
abstract: Asn185 is an invariant residue in all known sequences of TPL and of closely related tryptophanase and it may be aligned with the Asn194 in aspartate aminotransferase. According to X-ray data, in the holoenzyme and in the Michaelis complex Asn185 does not interact with the cofactor pyridoxal 5'-phosphate, but in the external aldimine a conformational change occurs which is accompanied by formation of a hydrogen bond between Asn185 and the oxygen atom in position 3 of the cofactor. The substitution of Asn185 in TPL by alanine results in a mutant N185A TPL of moderate residual activity (2%) with respect to adequate substrates, L-tyrosine and 3-fluoro-L-tyrosine. The affinities of the mutant enzyme for various amino acid substrates and inhibitors, studied by both steady-state and rapid kinetic techniques, were lower than for the wild-type TPL. This effect mainly results from destabilization of the quinonoid intermediate, and it is therefore concluded that the hydrogen bond between Asn185 and the oxygen at the C-3 position of the cofactor is maintained in the quinonoid intermediate. The relative destabilization of the quinonoid intermediate and external aldimine leads to the formation of large amounts of gem-diamine in reactions of N185A TPL with 3-fluoro-L-tyrosine and L-phenylalanine. For the reaction with 3-fluoro-L-tyrosine it was first possible to determine kinetic parameters of gem-diamine formation by the stopped-flow method. For the reactions of N185A TPL with substrates bearing good leaving groups the observed values of k(cat) could be accounted for by taking into consideration two effects: the decrease in the quinonoid content under steady-state conditions and the increase in the quinonoid reactivity in a beta-elimination reaction. Both effects are due to destabilization of the quinonoid and they counterbalance each other. Multiple kinetic isotope effect studies on the reactions of N185A TPL with suitable substrates, L-tyrosine and 3-fluoro-L-tyrosine, show that the principal mechanism of catalysis, suggested previously for the wild-type enzyme, does not change. In the framework of this mechanism the observed considerable decrease in k(cat) values for reactions of N185A TPL with L-tyrosine and 3-fluoro-L-tyrosine may be ascribed to participation of Asn185 in additional stabilization of the keto quinonoid intermediate.
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, Moscow 117984, Russia.
authors.forename: M V
authors.initials: MV
authors.lastname: Barbolina
authors.forename: R S
authors.initials: RS
authors.lastname: Phillips
authors.forename: P D
authors.initials: PD
authors.lastname: Gollnick
authors.forename: N G
authors.initials: NG
authors.lastname: Faleev
authors.forename: T V
authors.initials: TV
authors.lastname: Demidkina
mesh_terms: D000409:Alanine; D019943:Amino Acid Substitution; D001216:Asparagine; D001219:Aspartate Aminotransferases / Q000737:chemistry; D016971:Citrobacter freundii / Q000201:enzymology*; D006860:Hydrogen Bonding; D007700:Kinetics; D008962:Models, Theoretical; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011809:Quinones; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012984:Software; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D014447:Tyrosine Phenol-Lyase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D011809:Quinones; D011994:Recombinant Proteins; D014443:Tyrosine; D001216:Asparagine; D001219:Aspartate Aminotransferases; D014447:Tyrosine Phenol-Lyase; D000409:Alanine