Wild Markus R, Pos Klaas M, Dimroth Peter
Mikrobiologisches Institut der Eidgenössischen Technischen Hochschule, ETH-Zentrum, CH-8092 Zürich, Switzerland.
Biochemistry. 2003 Oct 14;42(40):11615-24. doi: 10.1021/bi034753k.
Helix VIII of the beta-subunit of the oxaloacetate decarboxylase of Klebsiella pneumoniae contains the functionally important residues betaN373, betaG377, betaS382, and betaR389. Using a functional oxaloacetate decarboxylase mutant devoid of Cys residues in the beta-subunit, each amino acid residue in helix VIII was replaced individually with Cys. Structural and dynamic features of this region were studied by using site-directed sulfhydryl modification of 20 single-Cys replacement mutants with methanethiosulfonate (MTS) reagents in the absence or presence of Na(+) ions. The pattern of accessibility of the MTS reagents from the periplasmic side of helix VIII shows a periodicity which suggests that this region is alpha-helical. In particular, a water-accessible face comprising betaN373, betaG377, betaS382, betaM386, and betaV390 may be part of a Na(+) channel. Cys residues introduced in the cytoplasmically oriented part of helix VIII were accessible to three different water-soluble MTS compounds and therefore believed to be exposed to water on this side of the membrane. Most residues located in the upper part of helix VIII (residues betaN373-betaV381C) were protected by Na(+) ions for inactivation by the MTS reagents. The distinct results on accessibility toward the different MTS reagents obtained in the presence or absence of Na(+) ions may suggest a conformational change upon binding of Na(+) in this region. The betaR389C mutant had a reduced activity and a pH optimum at pH 9, which could be restored to a wild-type pH optimum of 6.5 and to a 400% gain in activity upon chemical modification with 2-aminoethyl methanethiosulfonate.
肺炎克雷伯菌草酰乙酸脱羧酶β亚基的螺旋 VIII 包含功能上重要的残基βN373、βG377、βS382 和βR389。使用β亚基中不含半胱氨酸残基的功能性草酰乙酸脱羧酶突变体，将螺旋 VIII 中的每个氨基酸残基分别替换为半胱氨酸。通过在不存在或存在 Na⁺离子的情况下用甲硫代磺酸盐（MTS）试剂对 20 个单半胱氨酸替代突变体进行定点巯基修饰，研究了该区域的结构和动力学特征。MTS 试剂从螺旋 VIII 的周质侧的可及性模式显示出周期性，这表明该区域是α螺旋。特别是，由βN373、βG377、βS382、βM386 和βV390 组成的可被水接触的面可能是 Na⁺通道的一部分。引入螺旋 VIII 面向细胞质部分的半胱氨酸残基可被三种不同的水溶性 MTS 化合物接触，因此被认为在膜的这一侧暴露于水。位于螺旋 VIII 上部的大多数残基（残基βN373-βV381C）受到 Na⁺离子的保护，免受 MTS 试剂的失活作用。在存在或不存在 Na⁺离子的情况下获得的关于对不同 MTS 试剂可及性的不同结果可能表明该区域在结合 Na⁺时发生了构象变化。βR389C 突变体的活性降低，最适 pH 为 9，在用甲硫基乙磺酸盐进行化学修饰后，可恢复到野生型的最适 pH 6.5，并使活性提高 400%。
