title: Tryptophanase from Proteus vulgaris: the conformational rearrangement in the active site, induced by the mutation of Tyrosine 72 to phenylalanine, and its mechanistic consequences.
abstract: Tyr72 is located at the active site of tryptophanase (Trpase) from Proteus vulgaris. For the wild-type Trpase Tyr72 might be considered as the general acid catalyst at the stage of elimination of the leaving groups. The replacement of Tyr72 by Phe leads to a decrease in activity for L-tryptophan by 50,000-fold and to a considerable rearrangement of the active site of Trpase. This rearrangement leads to an increase of room around the alpha-C atom of any bound amino acid, such that covalent binding of alpha-methyl-substituted amino acids becomes possible (which cannot be realized in wild-type Trpase). The changes in reactivities of S-alkyl-L-cysteines provide evidence for an increase of congestion in the proximity of their side groups in the mutant enzyme as compared to wild-type enzyme. The observed alteration of catalytic properties in a large degree originates from a conformational change in the active site. The Y72F Trpase retains significant activity for L-serine, which allowed us to conclude that in the mutant enzyme, some functional group is present which fulfills the role of the general acid catalyst in reactions associated with elimination of small leaving groups.
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, Moscow 119991, Russia.
authors.forename: Vitalia V
authors.initials: VV
authors.lastname: Kulikova
authors.forename: Ludmila N
authors.initials: LN
authors.lastname: Zakomirdina
authors.forename: Irene S
authors.initials: IS
authors.lastname: Dementieva
authors.forename: Robert S
authors.initials: RS
authors.lastname: Phillips
authors.forename: Paul D
authors.initials: PD
authors.lastname: Gollnick
authors.forename: Tatyana V
authors.initials: TV
authors.lastname: Demidkina
authors.forename: Nicolai G
authors.initials: NG
authors.lastname: Faleev
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry*; D011487:Protein Conformation / Q000187:drug effects; D011514:Proteus vulgaris / Q000201:enzymology; D014368:Tryptophanase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D014443:Tyrosine; D010649:Phenylalanine; D014368:Tryptophanase