title: Crystals of tryptophan indole-lyase and tyrosine phenol-lyase form stable quinonoid complexes.
abstract: The binding of substrates and inhibitors to wild-type Proteus vulgaris tryptophan indole-lyase and to wild type and Y71F Citrobacter freundii tyrosine phenol-lyase was investigated in the crystalline state by polarized absorption microspectrophotometry. Oxindolyl-lalanine binds to tryptophan indole-lyase crystals to accumulate predominantly a stable quinonoid intermediate absorbing at 502 nm with a dissociation constant of 35 microm, approximately 10-fold higher than that in solution. l-Trp or l-Ser react with tryptophan indole-lyase crystals to give, as in solution, a mixture of external aldimine and quinonoid intermediates and gem-diamine and external aldimine intermediates, respectively. Different from previous solution studies (Phillips, R. S., Sundararju, B., & Faleev, N. G. (2000) J. Am. Chem. Soc. 122, 1008-1114), the reaction of benzimidazole and l-Trp or l-Ser with tryptophan indole-lyase crystals does not result in the formation of an alpha-aminoacrylate intermediate, suggesting that the crystal lattice might prevent a ligand-induced conformational change associated with this catalytic step. Wild-type tyrosine phenol-lyase crystals bind l-Met and l-Phe to form mixtures of external aldimine and quinonoid intermediates as in solution. A stable quinonoid intermediate with lambda(max) at 502 nm is accumulated in the reaction of crystals of Y71F tyrosine phenol-lyase, an inactive mutant, with 3-F-l-Tyr with a dissociation constant of 1 mm, approximately 10-fold higher than that in solution. The stability exhibited by the quinonoid intermediates formed both by wild-type tryptophan indole-lyase and by wild type and Y71F tyrosine phenol-lyase crystals demonstrates that they are suitable for structural determination by x-ray crystallography, thus allowing the elucidation of a key species of pyridoxal 5'-phosphate-dependent enzyme catalysis.
authors.affiliation: Department of Chemistry, University of Georgia, Athens, Georgia 30602, USA. rsphillips@chem.uga.edu
authors.forename: Robert S
authors.initials: RS
authors.lastname: Phillips
authors.forename: Tatyana V
authors.initials: TV
authors.lastname: Demidkina
authors.forename: Lyudmila N
authors.initials: LN
authors.lastname: Zakomirdina
authors.forename: Stefano
authors.initials: S
authors.lastname: Bruno
authors.forename: Luca
authors.initials: L
authors.lastname: Ronda
authors.forename: Andrea
authors.initials: A
authors.lastname: Mozzarelli
mesh_terms: D002384:Catalysis; D016971:Citrobacter freundii / Q000201:enzymology; D003460:Crystallization; D007700:Kinetics; D008956:Models, Chemical; D011485:Protein Binding; D011514:Proteus vulgaris / Q000201:enzymology; D012694:Serine / Q000737:chemistry; D013053:Spectrophotometry; D014368:Tryptophanase / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry; D014447:Tyrosine Phenol-Lyase / Q000737:chemistry* / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D014443:Tyrosine; D012694:Serine; D014368:Tryptophanase; D014447:Tyrosine Phenol-Lyase