Phillips Robert S, Demidkina Tatyana V, Zakomirdina Lyudmila N, Bruno Stefano, Ronda Luca, Mozzarelli Andrea
Department of Chemistry, University of Georgia, Athens, Georgia 30602, USA.
J Biol Chem. 2002 Jun 14;277(24):21592-7. doi: 10.1074/jbc.M200216200. Epub 2002 Apr 4.
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.
通过偏振吸收显微分光光度法，在晶体状态下研究了底物和抑制剂与野生型普通变形杆菌色氨酸吲哚裂解酶以及野生型和Y71F弗氏柠檬酸杆菌酪氨酸苯酚裂解酶的结合情况。氧化吲哚基丙氨酸与色氨酸吲哚裂解酶晶体结合，主要积累一种稳定的醌型中间体，其在502nm处有吸收，解离常数为35微摩尔，比在溶液中高约10倍。l-色氨酸或l-丝氨酸与色氨酸吲哚裂解酶晶体反应，与在溶液中一样，分别生成外部醛亚胺和醌型中间体的混合物以及偕二胺和外部醛亚胺中间体。与之前的溶液研究（菲利普斯，R.S.，桑达拉朱，B.，&法列耶夫，N.G.（2000）《美国化学会志》122，1008 - 1114）不同，苯并咪唑与l-色氨酸或l-丝氨酸与色氨酸吲哚裂解酶晶体的反应不会导致α-氨基丙烯酸酯中间体的形成，这表明晶格可能会阻止与该催化步骤相关的配体诱导的构象变化。野生型酪氨酸苯酚裂解酶晶体与l-甲硫氨酸和l-苯丙氨酸结合，形成与溶液中一样的外部醛亚胺和醌型中间体的混合物。在无活性突变体Y71F酪氨酸苯酚裂解酶晶体与3-F-l-酪氨酸的反应中，积累了一种λ(max)在502nm的稳定醌型中间体，解离常数为1毫摩尔，比在溶液中高约10倍。野生型色氨酸吲哚裂解酶以及野生型和Y71F酪氨酸苯酚裂解酶晶体形成的醌型中间体所表现出的稳定性表明，它们适合通过X射线晶体学进行结构测定，从而有助于阐明磷酸吡哆醛依赖性酶催化的关键物种。
