Griessler R, D'Auria S, Tanfani F, Nidetzky B
Division of Biochemical Engineering, Institute of Food Technology, Universität für Bodenkultur Wien (BOKU), Austria.
Protein Sci. 2000 Jun;9(6):1149-61. doi: 10.1110/ps.9.6.1149.
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 (> or = 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 Ksulfate = 4.5 mM and Kphosphate approximately 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 1H/2H 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 approximately 50 degrees C, and (2) stabilizes the covalent protein cofactor linkage up to approximately 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 approximately 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 approximately 20 degrees C higher value on the temperature scale.
来自卡氏棒状杆菌的淀粉磷酸化酶是一种二聚体蛋白，每摩尔90 kDa的亚基含有1摩尔吡哆醛5'-磷酸作为活性位点辅因子。为了确定磷酸根离子或硫酸根离子在高温（≥50℃）下使酶对不可逆失活具有大于500倍稳定性的机制，研究了酶与含氧阴离子的相互作用及其在磷酸化酶热变性过程中的作用。通过与一个不同于催化位点的蛋白质位点结合，硫酸根的解离常数Ksulfate = 4.5 mM，磷酸根的解离常数Kphosphate约为16 mM，二价含氧阴离子诱导磷酸化酶形成更紧密的结构，表现为：（a）α-螺旋二级结构的相对组成增加约5%；（b）1H/2H交换减少；（c）辅因子荧光受到保护，免受碘化物淬灭。酶活性的不可逆丧失是由吡哆醛5'-磷酸释放到溶液中引发的，是由磷酸化酶亚基之间的疏水相互作用驱动的分子间聚集导致的，这些亚基的二级结构呈现出温度依赖性的解链程度。通过特异性增加磷酸化酶二聚体结构的稳定性（可能是由于亚基间接触紧密），磷酸根和硫酸根间接（1）在高达约50℃时保持功能性活性位点，（2）在高达约70℃时稳定共价蛋白辅因子连接。对热稳定性的影响对磷酸根浓度呈现S形和饱和依赖性，在50℃时的表观结合常数约为25 mM。含氧阴离子-配体与淀粉磷酸化酶结合赋予的额外稳定性表现为整个变性途径在温度尺度上显著向约高20℃的值移动。
