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 phenollyase was investigated in the crystalline state by polarized absorption microspectrophotometry. Oxindolyl-L-alanine binds to tryptophan indole-lyase crystals to accumulate predominantly a stable quinonoid intermediate absorbing at 502 nm with a dissociation constant of 35 μM, 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 α-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 λ 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.