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首页> 外文期刊>Biochemistry >Mutational analysis of allylic substrate binding site of Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase.
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Mutational analysis of allylic substrate binding site of Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase.

机译：微球型B-P 26中未赤赤二磷酸二磷酸合酶烯丙基底物结合位点的突变分析。

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摘要

Undecaprenyl diphosphate (UPP) synthase catalyzes the sequential cis-condensation of isopentenyl diphosphate (IPP) onto (E,E)-farnesyl diphosphate (FPP). In our previous reports on the Micrococcus luteus B-P 26 UPP synthase, we have shown that the conserved residues in the disordered region from Ser-74 to Val-85 is crucial for the binding of FPP and the catalytic function [Fujikura, K., et al. (2000) J. Biochem. (Tokyo) 128, 917-922] and the existence of a structural P-loop motif for the FPP binding site [Fujihashi, M., et al. (2001) Proc. Natl. Acad. Sci. U.S.A., 98, 4337-4342]. To elucidate the allylic substrate binding site in more detail, we prepared eight mutant enzymes and examined their kinetic behavior. The mutant with respect to the two complementarily conserved Arg residues among the structural P-loop motif, G32R-R42G, retained the activity and showed product distribution pattern exactly similar to that of the wild-type, indicating that the complementarily conserved Arg is important for maintaining the catalytic function. Substitutions of Asp-29, Arg-33, or Arg-80 with Ala resulted in a large loss of enzyme activity, suggesting that these residues are essential for catalytic function. However, the K(m) values of these mutant enzymes for Z-GGPP, which is the first intermediate during the enzymatic cis-condensations of IPP onto FPP, were only moderately different or little changed from those of the wild type. These results suggest that the binding site for the intermediate Z-GGPP having a cis double bond is different to that for the intrinsic allylic substrate, FPP, whose diphosphate moiety is recognized by the structural P-loop.
机译：非赤烷基二磷酸（UPP）合成酶催化异戊基二磷酸二磷酸（IPP）的顺序顺式CIS-缩合（E，E） - 羟基二磷酸（FPP）。在我们之前关于Microcococcus BP 26 UPP合酶的报告中，我们已经表明，来自Ser-74至Val-85的无序区域中的保守残基对于FPP和催化功能的结合至关重要[Fujikura，K。等al。 （2000）J. Biochem。 （Tokyo）128,917-922]以及FPP结合位点的结构P环基序的存在[Fujihashi，M.等人。 （2001）Proc。 natl。阿卡。 SCI。 U.S.A.，98,4337-4342]。为了更详细地阐明烯丙基底物结合位点，我们制备了八个突变酶并检查了其动力学行为。突变体相对于两个互补的保守的蛋白残留物在结构p环矩阵，G32R-R42G，保留活性并显示出与野生型的产品分布图案完全相似，表明互补保守的arg是重要的保持催化功能。 ASP-29，ARG-33或ARG-80的取代导致酶活性大量损失，表明这些残留物对于催化功能是必不可少的。然而，对于Z-GGPP的这些突变酶的K（M）值，其是ICPP上的酶CIS-缩合期间的第一中间体，仅从野生型中的中度不同或更短。这些结果表明，具有顺式双键的中间Z-GGPP的结合位点与本征烯丙基衬底FPP的Z-GGPP的结合位点与其二磷酸酯的氨基磷酸盐部分通过结构p环识别。

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  • 来源
    《Biochemistry》 |2003年第14期|共7页
  • 作者

    Fujikura K; Zhang YW; Fujihashi M; Miki K; Koyama T;

  • 作者单位

    Institute of Multidisciplinary Research for Advanced Materials Tohoku University Katahira 2-1-1 Aoba-ku Sendai 980-8577 Japan.;

    The Jackson Laboratory 600 Main Street Bar Harbor ME 04609 USA;

    The Jackson Laboratory 600 Main Street Bar Harbor ME 04609 USA;

    The Jackson Laboratory 600 Main Street Bar Harbor ME 04609 USA;

    The Jackson Laboratory 600 Main Street Bar Harbor ME 04609 USA;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

    Binding Sites; Arginine; P-2; mutant; 结合部位; 精氨酸;

    机译：Binding Sites;Arginine;P-2;mutant;结合部位;精氨酸;

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