Kursula I, Salin M, Sun J, Norledge B V, Haapalainen A M, Sampson N S, Wierenga R K
Department of Biochemistry and Biocenter Oulu, University of Oulu, PO Box 3000, FIN-90014 University of Oulu, Finland.
Protein Eng Des Sel. 2004 Apr;17(4):375-82. doi: 10.1093/protein/gzh048. Epub 2004 May 27.
The conformational switch from open to closed of the flexible loop 6 of triosephosphate isomerase (TIM) is essential for the catalytic properties of TIM. Using a directed evolution approach, active variants of chicken TIM with a mutated C-terminal hinge tripeptide of loop 6 have been generated (Sun,J. and Sampson,N.S., Biochemistry, 1999, 38, 11474-11481). In chicken TIM, the wild-type C-terminal hinge tripeptide is KTA. Detailed enzymological characterization of six variants showed that some of these (LWA, NPN, YSL, KTK) have decreased catalytic efficiency, whereas others (KVA, NSS) are essentially identical with wild-type. The structural characterization of these six variants is reported. No significant structural differences compared with the wild-type are found for KVA, NSS and LWA, but substantial structural adaptations are seen for NPN, YSL and KTK. These structural differences can be understood from the buried position of the alanine side chain in the C-hinge position 3 in the open conformation of wild-type loop 6. Replacement of this alanine with a bulky side chain causes the closed conformation to be favored, which correlates with the decreased catalytic efficiency of these variants. The structural context of loop 6 and loop 7 and their sequence conservation in 133 wild-type sequences is also discussed.
磷酸丙糖异构酶（TIM）柔性环6从开放构象到闭合构象的转变对于TIM的催化特性至关重要。利用定向进化方法，已产生了鸡TIM的活性变体，其环6的C端铰链三肽发生了突变（Sun，J.和Sampson，N.S.，《生物化学》，1999年，38卷，11474 - 11481页）。在鸡TIM中，野生型C端铰链三肽为KTA。对六个变体进行的详细酶学表征表明，其中一些（LWA、NPN、YSL、KTK）催化效率降低，而其他一些（KVA、NSS）与野生型基本相同。本文报道了这六个变体的结构表征。KVA、NSS和LWA与野生型相比未发现明显的结构差异，但NPN、YSL和KTK则出现了显著的结构适应性变化。这些结构差异可以从野生型环6开放构象中丙氨酸侧链在C铰链位置3的埋藏位置来理解。用一个大的侧链取代这个丙氨酸会使闭合构象更受青睐，这与这些变体催化效率的降低相关。本文还讨论了环6和环7的结构背景及其在133个野生型序列中的序列保守性。
