Ngo Huu, Kimmich Novelle, Harris Rodney, Niks Dimitri, Blumenstein Lars, Kulik Victor, Barends Thomas Reinier, Schlichting Ilme, Dunn Michael F
Department of Biochemistry, University of California, Riverside, California 92521, USA.
Biochemistry. 2007 Jul 3;46(26):7740-53. doi: 10.1021/bi7003872. Epub 2007 Jun 9.
In the tryptophan synthase bienzyme complex, indole produced by substrate cleavage at the alpha-site is channeled to the beta-site via a 25 A long tunnel. Within the beta-site, indole and l-Ser react with pyridoxal 5'-phosphate in a two-stage reaction to give l-Trp. In stage I, l-Ser forms an external aldimine, E(Aex1), which converts to the alpha-aminoacrylate aldimine, E(A-A). Formation of E(A-A) at the beta-site activates the alpha-site >30-fold. In stage II, indole reacts with E(A-A) to give l-Trp. The binding of alpha-site ligands (ASLs) exerts strong allosteric effects on the reaction of substrates at the beta-site: the distribution of intermediates formed in stage I is shifted in favor of E(A-A), and the binding of ASLs triggers a conformational change in the beta-site to a state with an increased affinity for l-Ser. Here, we compare the behavior of new ASLs as allosteric effectors of stage I with the behavior of the natural product, d-glyceraldehyde 3-phosphate. Rapid kinetics and kinetic isotope effects show these ASLs bind with affinities ranging from micro- to millimolar, and the rate-determining step for conversion of E(Aex1) to E(A-A) is increased by 8-10-fold. To derive a structure-based mechanism for stage I, X-ray structures of both the E(Aex1) and E(A-A) states complexed with the different ASLs were determined and compared with structures of the ASL complexes with the internal aldimine [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Biochemistry 46, 7713-7727].
在色氨酸合酶双酶复合物中，α位点底物裂解产生的吲哚通过一条25埃长的通道输送到β位点。在β位点，吲哚和L-丝氨酸与磷酸吡哆醛发生两步反应生成L-色氨酸。在第一步中，L-丝氨酸形成外部醛亚胺E(Aex1)，其转化为α-氨基丙烯酸醛亚胺E(A-A)。在β位点形成E(A-A)会使α位点的活性提高30倍以上。在第二步中，吲哚与E(A-A)反应生成L-色氨酸。α位点配体（ASLs）的结合对β位点底物的反应产生强烈的变构效应：第一步形成的中间体分布向有利于E(A-A)的方向移动，ASLs的结合引发β位点的构象变化，使其对L-丝氨酸的亲和力增加。在此，我们将新型ASLs作为第一步变构效应剂的行为与天然产物3-磷酸-D-甘油醛的行为进行了比较。快速动力学和动力学同位素效应表明，这些ASLs的结合亲和力范围为微摩尔至毫摩尔，E(Aex1)转化为E(A-A)的速率决定步骤增加了8至10倍。为了推导第一步基于结构的机制，我们测定了与不同ASLs复合的E(Aex1)和E(A-A)状态的X射线结构，并将其与ASL与内部醛亚胺的复合物结构进行了比较[Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Biochemistry 46, 7713 - 7727]。
