Thermal denaturation pathway of starch phosphorylase from Corynebacterium callunae:: Oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the protein
Thermal denaturation pathway of starch phosphorylase from Corynebacterium callunae:: Oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the protein
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DOI：
10.1110/ps.9.6.1149
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发表时间：
2000-06-01
期刊：
影响因子：
8
通讯作者：
Nidetzky, B
中科院分区：
文献类型：
--
作者：
Griessler, R;D'Auria, S;Nidetzky, B
愈伤棒状杆菌的淀粉磷酸化酶是一种二聚体蛋白，每90 kDa亚基中含有1摩尔的吡哆醛5‘-磷酸作为活性部位的辅因子。为了确定磷酸盐或硫酸盐离子在高温(大于或等于50℃)下对不可逆失活产生500倍以上的稳定性的机制，研究了酶/氧阴离子相互作用及其在磷酸化酶热变性中的作用。通过与解离常数分别为K-硫酸盐=4.5 mM和K-磷酸接近16 mM的催化位区分的蛋白质位置，二阴离子氧阴离子诱导形成更紧密的磷酸化结构，表现为：(A)α-螺旋二级结构的相对组成增加约5%，(B)减少H-1/H-2交换，以及(C)保护辅因子荧光免受碘的猝灭。不可逆的酶活性丧失是由5‘-磷酸吡哆醛释放到溶液中引发的，是由磷酸化酶亚基之间的疏水作用驱动的后续分子间聚集导致的，这种聚集表现出二级结构的熔融程度与温度有关。通过特别增加磷酸化酶(可能是由于亚基间接触紧密)、磷酸和硫酸盐的二聚体结构的稳定性，这间接地(1)保留了高达约50摄氏度的功能性活性部位，(2)稳定了高达约70摄氏度的共价蛋白质辅因子连接。对热稳定性的影响显示出对磷酸盐浓度的S形和可饱和的依赖关系，在50摄氏度时的表观结合常数约为25 mm。氧阴离子配体与淀粉磷酸化酶结合所赋予的额外稳定性表现为整个变性途径的戏剧性转变，在温度标度上大约高出20摄氏度。
Starch phosphorylase from Corynebacterium callunae is a dimeric protein in which each mol of 90 kDa subunit contains 1 mol pyridoxal 5'-phosphate as an active-site cofactor. To determine the mechanism by which phosphate or sulfate ions bring about a greater than 500-fold stabilization against irreversible inactivation at elevated temperatures (greater than or equal to 50 degrees C), enzyme/oxyanion interactions and their role during thermal denaturation of phosphorylase have been studied. By binding to a protein site distinguishable from the catalytic site with dissociation constants of K-sulfate = 4.5 mM and K-phosphate approximate to 16 mM, dianionic oxyanions induce formation of a more compact structure of phosphorylase, manifested by (a) an increase by about 5% in the relative composition of the alpha-helical secondary structure, (b) reduced H-1/H-2 exchange, and (c) protection of a cofactor fluorescence against quenching by iodide. irreversible loss of enzyme activity is triggered by the release into solution of pyridoxal 5'-phosphate, and results from subsequent intermolecular aggregation driven by hydrophobic interactions between phosphorylase subunits that display a temperature-dependent degree of melting of secondary structure. By specifically increasing the stability of the dimer structure of phosphorylase (probably due to tightened intersubunit contacts), phosphate, and sulfate, this indirectly (1) preserves a functional active site up to approximate to 50 degrees C, and (2) stabilizes the covalent protein cofactor linkage up to approximate to 70 degrees C. The effect on thermostability shows a sigmoidal and saturatable dependence on the concentration of phosphate, with an apparent binding constant at 50 degrees C of approximate to 25 mM. The extra stability conferred by oxyanion-ligand binding to starch phosphorylase is expressed as a dramatic shift of the entire denaturation pathway to a approximate to 20 degrees C higher value on the temperature scale.