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Mechanistic studies of human spermine oxidase: Kinetic mechanism and pH effects

机译：人精灵氧化酶的机械研究：动力学机制和pH效应

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摘要

In mammalian cells, the flavoprotein spermine oxidase (SMO) catalyzes the oxidation of spermine to spermidine and 3-aminopropanal. Mechanistic studies have been conducted with the recombinant human enzyme. The initial velocity pattern in which the ratio between the concentrations of spermine and oxygen is kept constant establishes the steady-state kinetic pattern as ping-pong. Reduction of SMO by spermine in the absence of oxygen is biphasic. The rate constant for the rapid phase varies with the substrate concentration, with a limiting value (k_3) of 49 s~(-1) and an apparent K d value of 48 μMat pH8.3. The rate constant for the slow step is independent of the spermine concentration, with a value of 5.5 s~(-1), comparable to the k_(cat) value of 6.6 s~(-1). The kinetics of the oxidative half-reaction depend on the aging time after the spermine and enzyme are mixed in a double-mixing experiment. At an aging time of 6 s, the reaction is monophasic with a second-order rate constant of 4.2 mM~(-1) s~(-1). At an aging time of 0.3 s, the reaction is biphasic with two second-order constants equal to 4.0 and 40 mM~(-1) s~(-1). Neither is equal to the k_(cat)/K_(O2) value of 13 mM -1 s~(-1). These results establish the existence of more than one pathway for the reaction of the reduced flavin intermediate with oxygen. The k_(cat)/KM value for spermine exhibits a bell-shaped pH profile, with an average pKa value of 8.3. This profile is consistent with the active form of spermine having three charged nitrogens. The pH profile for k_3 shows a pK_a value of 7.4 for a group that must be unprotonated. The pK_i-pH profiles for the competitive inhibitors N,N′-dibenzylbutane-1,4-diamine and spermidine show that the fully protonated forms of the inhibitors and the unprotonated form of an amino acid residue with a pK_a of ~7.4 in the active site are preferred for binding.
机译：在哺乳动物细胞中，黄酮蛋白精蛋白氧化酶（Smo）催化精霉素氧化至亚精胺和3-氨基丙烷。用重组人酶进行机械研究。初始速度模式，其中浓度和氧浓度与氧气之间的比率保持恒定地建立稳态动力学图案作为平乒乓。在没有氧气的情况下，精美通过精度减少了双相。快速相的速率常数随底物浓度而变化，限制值（K_3）为49s〜（-1），表观k d值48μmatpH8.3。慢步的速率常数与精素浓度无关，值为5.5 s〜（-1），可与6.6 s〜（-1）的k_（猫）值相当。氧化半反应的动力学取决于在双混合实验中混合精霉和酶后的老化时间。在6 s的老化时间下，反应是单相表格，其二阶速率常数为4.2mm〜（-1）s〜（-1）。在0.3秒的老化时间下，反应是双相，其两顺常数等于4.0和40mm〜（-1）S〜（-1）。两者都不等于K_（CAT）/ k_（o2）值为13 mm -1 s〜（-1）。这些结果建立了不止一种途径的存在，用于减少黄素中间体与氧气的反应。 Femermine的K_（猫）/ km值表现出钟形pH型材，平均pKa值为8.3。该概况与具有三个带电氮的活性形式的精霉素一致。用于K_3的pH分布显示了必须不普遍的组的PK_A值为7.4。竞争性抑制剂N，N'-二苄基丁烷-1,4-二胺和亚氨基的PK_I-pH分布表明，抑制剂的完全质子化形式和在活性的PK_A的氨基酸残基的非普促形式的形式遗址优选结合。

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  • 来源
    《Biochemistry》 |2010年第2期|共7页
  • 作者

    Adachi M.S.; Juarez P.R.; Fitzpatrick P.F.;

  • 作者单位

    Department of Biochemistry Center for Biomedical Neuroscience University of Texas Health Science Center San Antonio TX 78229 United States;

    Department of Biochemistry and Biophysics Texas A and M University College Station TX 77843 United States;

    Department of Biochemistry Center for Biomedical Neuroscience University of Texas Health Science Center San Antonio TX 78229 United States;

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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

    Mechanistic studies; human spermine; Kinetic mechanism;

    机译：机械研究;人精灵;动力学机制;

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