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.
Highlights
Tyrosine phenol-lyase (TPL)1 (EC 4.1.99.2) and tryptophan indole-lyase (Trpase, EC 4.1.99.1) are pyridoxal 5Ј-phosphate (PLP)-dependent enzymes that catalyze -elimination reactions to form phenol or indole and ammonium pyruvate from tyrosine and tryptophan, respectively (Schemes 1 and 2) [1, 2]
An addition of oxindolyl-L-alanine (Scheme 4, I), a potent inhibitor of the E. coli enzyme [16], to crystals of P. vulgaris Trpase results in the formation of an intense absorbance peak at 502 nm that is attributed to a quinonoid intermediate (Fig. 1)
When P. vulgaris Trpase crystals are suspended in a solution containing 10 mM L-Trp, the polarized absorption spectra exhibit peaks at 425 and 505 nm with similar intensity corresponding to external aldimine and quinonoid intermediates, respectively (Fig. 2, a and b)
Summary
Materials—L-Tyr and L-Ser were obtained from Sigma. Benzimidazole and indole were purchased from Aldrich. 3-Fluoro-L-tyrosine was prepared from 2-fluorophenol and ammonium pyruvate using TPL as described previously [26]. Purification of Enzymes—E. coli SVS370 cells containing pTZTPL with wild type and the Y71F mutant TPL genes were grown, and the enzyme was purified as described previously [27]. Crystallization of TPL and Trpase—Crystallization of both wild-type TPL and Y71F mutant enzymes was performed with monomethyl ether polyethylene glycol 5000 (Fluka) by the hanging drop vapor diffusion technique using the conditions for the crystallization of the TPL complex with 3-(4Ј-hydroxyphenyl)propionic acid [15]. 2 l of a solution containing 20 mg/ml enzyme, 50 mM triethanolamine-HCl buffer, 0.2 mM PLP, 0.5 mM dithiothreitol, 0.1 M KCl, or 0.1 M CsCl, pH 8.0, were mixed with an equal volume of a reservoir solution. The best crystals were obtained using a solution containing 40 – 45% (w/v) monomethyl ether polyethylene glycol and either 0.1– 0.4 M KCl or 0.1– 0.4 M CsCl. 2 L.
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