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首页> 外文期刊>Biochemistry >Substrate Specificity Characterization of Recombinant Metall Oligo-Peptidases Thimet Oligopeptidase and Neurolysin
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Substrate Specificity Characterization of Recombinant Metall Oligo-Peptidases Thimet Oligopeptidase and Neurolysin

机译：重组金属寡肽酶的底物特异性表征紫藤寡肽酶和神经溶素

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摘要

We report a systematic and detailed analysis of recombinant neurolysin (BC 3.4.24.16) specificity in parallel with thimet oligopeptidase (TOP, BC 3.4.24.15) using Bk sequence and its C- and N-terminal extensions as in hum~1n kininogen as motif for synthesis of internally quenched fluorescent substrates. The influence of the substrate size was investigated, and the longest peptide susceptible to TOP and neurolysin contains 17 amino acids. The specificities of both oligopeptidases to substrate sites P4 to P3’ were also characterized in great detail using seven series of peptides based on Abz-GFSPFRQ-EDDnp taken as reference substrate. Most of the peptides were hydrolyzed at the bond corresponding to P4—F5 in the reference substrate and some of them were hydrolyzed at this bond or at F2—53 bond. No restricted specificity was found for P1’ as found in thermolysin as well for P1 substrate position, however the modifications at this position (P1) showed to have large influence on the catalytic constant and the best substrates for TOP contained at P1, Phe, Ala, or Arg and for neurolysin Asn or Arg. Some amino acid residues have large influence on the Km constants independently of its position. On the basis of these results, we are hypothesizing that some amino acids of the substrates can bind to different sub-sites of the enzyme fitting P—F or F—S bond, which requires rapid interchange for the different forms of interaction and convenient conformations of the substrate in order to expose and fit the cleavage bonds in correct position for an efficient hydrolysis. Finally, this plasticity of interaction with the substrates can be an essential property for a class of cytosolic oligopeptidases that are candidates to participate in the selection of the peptides to be presented by the MHC class I.
机译：我们通过使用BK序列及其C-和N-末端延伸，对重组神经蛋白（BC 3.4.24.16）的特异性进行了对重组神经蛋白酶（BC 3.4.24.16）的系统和详细分析，如HUM〜1N活性为主题用于合成内部淬火荧光基材。研究了基板尺寸的影响，并且易受顶部和神经蛋白的最长肽含有17个氨基酸。使用基于ABZ-GFSPFRQ-EDDNP的7系列肽作为参考衬底，使用基于ABZ-GFSPFRQ-EDDDNP的七系列肽的详细描述了对底物位点P4至P3'的特征。大多数肽在对应于参考底物中对应于P4-F5的键处水解，并且它们中的一些在该键或在F2-53键处水解。在P1底物位置中发现的P1'没有发现P1'的限制特异性，然而该位置（P1）的修饰显示出对催化恒定和最佳基材的催化常数和最佳基材的含量较大，或arg和神经溶素Asn或arg。一些氨基酸残基对km常量的影响大幅影响，其位置是其位置。在这些结果的基础上，我们已经假设底物的一些氨基酸可以与酶配合P-F或F-S键的不同子位点结合，这需要快速交换不同形式的相互作用和方便的构象基材以便在正确的位置暴露和适合裂解键，以获得有效的水解。最后，这种与基材的相互作用的可塑性可以是一类细胞溶质寡肽酶的基本性质，所述细胞溶质寡肽酶是参与由MHC级I呈现的肽的选择的候选物。

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  • 来源
    《Biochemistry》 |2001年第14期|共9页
  • 作者

    Vitor Oliveira; Marcelo Campos; Robson L. Melo; Emer S. Ferro; Antonino C. M. Camargo; Maria A. Juliano; Luiz Juliano;

  • 作者单位

    Department of Biophysics Escola Paulista de Medicina Universitdade Federal de Sao Paulo Rua Tres de Maio 100 Sao Paulo - SP - 04044-020 Brazil Department of Histology Institute of Biomedical Sciences Universidade de Sao Paulo Sao Paulo. 05508-900;

    Department of Biophysics Escola Paulista de Medicina Universitdade Federal de Sao Paulo Rua Tres de Maio 100 Sao Paulo - SP - 04044-020 Brazil Department of Histology Institute of Biomedical Sciences Universidade de Sao Paulo Sao Paulo. 05508-900;

    Department of Biophysics Escola Paulista de Medicina Universitdade Federal de Sao Paulo Rua Tres de Maio 100 Sao Paulo - SP - 04044-020 Brazil Department of Histology Institute of Biomedical Sciences Universidade de Sao Paulo Sao Paulo. 05508-900;

    Department of Biophysics Escola Paulista de Medicina Universitdade Federal de Sao Paulo Rua Tres de Maio 100 Sao Paulo - SP - 04044-020 Brazil Department of Histology Institute of Biomedical Sciences Universidade de Sao Paulo Sao Paulo. 05508-900;

    Department of Biophysics Escola Paulista de Medicina Universitdade Federal de Sao Paulo Rua Tres de Maio 100 Sao Paulo - SP - 04044-020 Brazil Department of Histology Institute of Biomedical Sciences Universidade de Sao Paulo Sao Paulo. 05508-900;

    Department of Biophysics Escola Paulista de Medicina Universitdade Federal de Sao Paulo Rua Tres de Maio 100 Sao Paulo - SP - 04044-020 Brazil Department of Histology Institute of Biomedical Sciences Universidade de Sao Paulo Sao Paulo. 05508-900;

    Department of Biophysics Escola Paulista de Medicina Universitdade Federal de Sao Paulo Rua Tres de Maio 100 Sao Paulo - SP - 04044-020 Brazil Department of Histology Institute of Biomedical Sciences Universidade de Sao Paulo Sao Paulo. 05508-900;

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  • 正文语种 eng
  • 中图分类 生物化学;
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