Takahashi N, Kakinuma H, Liu L, Nishi Y, Fujii I
Laboratory of Life Science and Biomolecular Engineering, Japan Tobacco, Inc., 6-2, Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512, Japan.
Nat Biotechnol. 2001 Jun;19(6):563-7. doi: 10.1038/89320.
Enzymes have evolved their ability to use binding energies for catalysis by increasing the affinity for the transition state of a reaction and decreasing the affinity for the ground state. To evolve abzymes toward higher catalytic activity, we have reconstructed an enzyme-evolutionary process in vitro. Thus, a phage-displayed combinatorial library from a hydrolytic abzyme, 6D9, generated by the conventional in vivo method with immunization of the transition-state analog (TSA), was screened against a newly devised TSA to optimize the differential affinity for the transition state relative to the ground state. The library format successfully afforded evolved variants with 6- to 20-fold increases in activity (kcat) as compared with 6D9. Structural analysis revealed an advantage of the in vitro evolution over the in vivo evolution: an induced catalytic residue in the evolved abzyme arises from double mutations in one codon, which rarely occur in somatic hypermutation in the immune response.
酶通过增强对反应过渡态的亲和力并降低对基态的亲和力，进化出了利用结合能进行催化的能力。为了使抗体酶具有更高的催化活性，我们在体外重建了一个酶进化过程。因此，针对一种新设计的过渡态类似物（TSA），筛选了一个通过用过渡态类似物免疫的传统体内方法产生的水解抗体酶6D9的噬菌体展示组合文库，以优化相对于基态对过渡态的差异亲和力。与6D9相比，该文库形式成功地获得了活性（kcat）提高6至20倍的进化变体。结构分析揭示了体外进化相对于体内进化的一个优势：进化后的抗体酶中一个诱导的催化残基源自一个密码子中的双重突变，这在免疫反应的体细胞超突变中很少发生。
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