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首页> 外文期刊>Biochemistry >Substrate-Assisted Movement of the Catalytic Lys 215 during Domain Closure:Site-Directed Mutagenesis Studies of Human 3-Phosphoglycerate Kinase
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Substrate-Assisted Movement of the Catalytic Lys 215 during Domain Closure:Site-Directed Mutagenesis Studies of Human 3-Phosphoglycerate Kinase

机译：催化Lys 215在结构域闭合期间的底物辅助运动：人3-磷酸糖激酶的现场定向诱变研究

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摘要

3-Phosphoglycerate kinase (PGK) is a two-domain hinge-bending enzyme.It is still unclear how the geometry of the active site is formed during domain closure and how the catalytic residues are brought into the optimal position for the reaction.Comparison of the three-dimensional structures in various open and closed conformations suggests a large (10 A) movement of Lys 215 during domain closure.This change would be required for direct participation of this side chain in both the catalyzed phospho transfer and the special anion-caused activation.To test the multiple roles of Lys 215,two mutants (K215A and K215R) were constructed from human PGK and characterized in enzyme kinetic and substrate binding studies.For comparison,mutants (R38A and R38K) of the known essential residue,Arg 38,were also produced.Drastic decreases (1500- and 500-fold,respectively),as in the case of R38A,were observed in the k_(cat) values of mutants K215A and K215R,approving the essential catalytic role of Lys 215.In contrast,the R38K mutation caused an only 1.5-fold decrease in activity.This emphasizes the importance of a very precise positioning of Lys 215 in the active site,in addition to its positive charge.The side chain of Lys 215 is also responsible for the substrate and anion-dependent activation,since these properties are abolished upon mutation.Among the kinetic constants mainly the K_m values of MgATP and 1,3-BPG are increased (approx 20- and approx 8-fold,respectively) in the case of the neutral K215A mutant,evidence of the interaction of Lys 215 with the transferring phospho group in the functioning complex.Weakening of MgATP binding (a moderate increase in K_d),but not of MgADP binding,upon mutation indicates an initial weak interaction of Lys 215 with the gamma-phosphate already in the nonfunctioning open conformation.Thus,during domain closure,Lys 215 possibly moves together with the transferring phosphate;meanwhile,this group is being positioned properly for catalysis.
机译：3-磷酸​​甘油激酶（PGK）是双域铰链弯曲酶。仍不清楚在结构域闭合期间形成活性位点的几何形状以及如何将催化残余物进入反应的最佳位置。各种开放和闭合构象中的三维结构表明了域闭合期间Lys 215的大（10A）运动。这种变化是在催化的磷酸转移和特殊阴离子引起的副链中直接参与这种变化。激活。要测试Lys 215的多种作用，从人PGK构建了两个突变体（K215A和K215R），其特征在于酶动力学和底物结合研究。已知必需残留物的比较，突变体（R38A和R38K），ARG 38也是产生的。在突变体K215A和K215R的K_（猫）值中观察到R38a的情况下，在突变体K215A和K215R的k_（猫）值中，批准Lys 215的必要催化作用，观察到r38a的速度降低（分别为1500-和500倍）。相反，R38K突变引起的活性仅为1.5倍。这强调了Lys 215在活性位点中的非常精确定位的重要性，除了其正电荷之外。Lys 215的侧链也负责基材和阴离子依赖性活化，因为这些性质在突变时被废除。动力学常数主要是MgATP和1,3-BPG的K_M值（分别为20-和大约8倍）中性K215a突变体，Lys 215与转移磷酸的相互作用的证据在运作复合物中。突变时，突变时，突变的MgATP结合（K_D中的中等增加）的展示表明Lys 215的初始弱相互作用表示Lys 215的初始弱相互作用。对于已经在非障碍开放构象中的γ-磷酸盐。在结构域闭合期间，Lys 215可能与转移磷酸盐一起移动;同时，该组正正确定位用于催化。

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  • 来源
    《Biochemistry》 |2005年第51期|共13页
  • 作者

    Beata Flachner; Andrea Varga; Judit Szabo; Laszlo Bama; Istvan Hajdii;

  • 作者单位

    Institute of Enzymology Biological Research Center Hungarian Academy of Sciences H-1518 Budapest P.O.Box 7 Hungary;

    Institute of Enzymology Biological Research Center Hungarian Academy of Sciences H-1518 Budapest P.O.Box 7 Hungary;

    Institute of Enzymology Biological Research Center Hungarian Academy of Sciences H-1518 Budapest P.O.Box 7 Hungary;

    Institute of Enzymology Biological Research Center Hungarian Academy of Sciences H-1518 Budapest P.O.Box 7 Hungary;

    Institute of Enzymology Biological Research Center Hungarian Academy of Sciences H-1518 Budapest P.O.Box 7 Hungary;

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  • 正文语种 eng
  • 中图分类 生物化学;
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