Mutagenesis of the dimer interface region of Corynebacterium callunae starch phosphorylase perturbs the phosphate-dependent conformational relay that enhances oligomeric stability of the enzyme
Mutagenesis of the dimer interface region of Corynebacterium callunae starch phosphorylase perturbs the phosphate-dependent conformational relay that enhances oligomeric stability of the enzyme
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DOI：
10.1093/jb/mvg178
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发表时间：
2003-10-01
影响因子：
2.7
通讯作者：
Tanfani, F
中科院分区：
文献类型：
--
作者：
Nidetzky, B;Griessler, R;Tanfani, F
我们利用丙氨酸扫描定点突变方法，对愈伤棒状杆菌的淀粉磷酸化酶二聚体接触区进行了定点突变，以探讨磷酸盐结合引起的蛋白质构象变化与该酶功能四级结构在磷酸盐存在时的500倍动力学稳定性之间的关系。在室温和热变性条件下，用傅立叶变换红外光谱(FT-IR)和酶活性测定对纯化的突变体(Ser-224、Arg-226、Arg-234和Arg-242)进行了表征。野生型和突变体在21120溶剂中的不同FT-IR光谱显示，在约1,550 cm(-1)处残留的酰胺II带强度变化很小，表明野生型的H-1/H-2交换明显受到突变的干扰。与野生型相比，H-1/H-2交换减少表明在S224A、R234A和R242A突变体中形成了更紧密的蛋白质结构，并与45℃下的不可逆热变性速率有关，这三个突变体的不可逆热变性速率比野生型低10倍。相比之下，突变体R226A在45℃的失活速度是野生型的2.5倍，并表现出更高的H-1/H-2交换。磷酸盐(20 MM)引起野生型的FT-IR光谱比S224A和234A突变体更大的变化，并导致野生型的稳定性比两个突变体高5倍。因此，在S224A和R234A突变体中，导致野生型淀粉磷酸化酶动力学稳定性的结构效应是部分互补的。用红外光谱分析比较了突变体和野生型在没有和存在稳定氧阴离子的情况下的热变性。在S224A和R234A突变体中，未连接野生型在40-50℃范围内的广泛变性转变减少，这主要反映了变性开始向高4-5℃转变。
We have used alanine-scanning site-directed mutagenesis of the dimer contact region of starch phosphorylase from Corynebacterium callunae to explore the relationship between a protein conformational change induced by phosphate binding and the up to 500-fold kinetic stabilization of the functional quarternary structure of this enzyme when phosphate is present. Purified mutants (at positions Ser-224, Arg-226, Arg-234, and Arg-242) were characterized by Fourier transform-infrared (FT-IR) spectroscopy and enzyme activity measurements at room temperature and under conditions of thermal denaturation. Difference FT-IR spectra of wild type and mutants in 21120 solvent revealed small changes in residual amide II band intensities at approximate to 1,550 cm(-1), indicating that H-1/H-2 exchange in the wild type is clearly perturbed by the mutations. Decreased H-1/H-2 exchange in comparison to wild type suggests formation of a more compact protein structure in S224A, R234A, and R242A mutants and correlates with rates of irreversible thermal denaturation at 45degreesC that are up to 10-fold smaller for the three mutants than the wild type. By contrast, the mutant R226A inactivates 2.5-fold faster at 45degreesC and shows a higher H-1/H-2 exchange than the wild type. Phosphate (20 mM) causes a greater change in FT-IR spectra of the wild type than in those of S224A and 234A mutants and leads to a 5-fold higher stabilization of the wild type than the two mutants. Therefore, structural effects of phosphate binding leading to kinetic stability of wild-type starch phosphorylase are partially complemented in the S224A and R234A mutants. Infrared spectroscopic measurements were used to compare thermal denaturations of the mutants and the wild type in the absence and presence of stabilizing oxyanion. The broad denaturation transition of unliganded wild type in the range 40-50degreesC is reduced in the S224A and R234A mutants, and this reflects mainly a shift of the onset of denaturation to a 4-5degreesC higher value.