Ahmad A, Akhtar M S, Bhakuni V
Division of Membrane Biology, Central Drug Research Institute, Lucknow 226 001, India.
Biochemistry. 2001 Feb 20;40(7):1945-55. doi: 10.1021/bi001933a.
Glucose oxidase (GOD) from Aspergillus niger is an acidic dimeric enzyme having a high degree of localization of negative charges on the enzyme surface and dimer interface. We have studied the effect of monovalent cations on the structure and stability of GOD using various optical spectroscopic techniques, limited proteolysis, size exclusion chromatography, differential scanning calorimetry, and enzymic activity measurements. The monovalent cations were found to influence the enzymic activity and tertiary structure of GOD, but no effect on the secondary structure of the enzyme was observed. The monovalent cation-stabilized GOD was found to have a more compact dimeric structure but lower enzymic activity than the native enzyme. The enzyme's K(m) for D-glucose was found to be slightly enhanced for the monovalent cation-stabilized enzyme (maximum enhancement of about 35% for LiCl) as compared to native GOD. Comparative denaturation studies on the native and monovalent cation-stabilized enzyme demonstrated a significant resistance of cation-stabilized GOD to urea (about 50% residual activity at 6.5 M urea) and thermal denaturation (Delta T(m) maximum of 10 degrees C compared to native enzyme). However, pH-induced denaturation showed a destabilization of monovalent cation-stabilized GOD as compared to the native enzyme. The effectiveness of monovalent cations in stabilizing GOD structure against urea and thermal denaturation was found to follow the Hofmeister series: K(+) > Na(+) > Li(+).
黑曲霉葡萄糖氧化酶（GOD）是一种酸性二聚体酶，其酶表面和二聚体界面带有高度局部化的负电荷。我们使用各种光学光谱技术、有限蛋白酶解、尺寸排阻色谱、差示扫描量热法和酶活性测量等方法，研究了单价阳离子对GOD结构和稳定性的影响。发现单价阳离子会影响GOD的酶活性和三级结构，但未观察到对该酶二级结构的影响。发现单价阳离子稳定的GOD具有比天然酶更紧密的二聚体结构，但酶活性较低。与天然GOD相比，单价阳离子稳定的酶对D-葡萄糖的K(m)略有增加（LiCl时最大增加约35%）。对天然酶和单价阳离子稳定酶的比较变性研究表明，阳离子稳定的GOD对尿素具有显著抗性（在6.5 M尿素时约有50%的残余活性），对热变性也有显著抗性（与天然酶相比，ΔT(m)最大为10℃）。然而，与天然酶相比，pH诱导的变性表明单价阳离子稳定的GOD不稳定。发现单价阳离子在稳定GOD结构以抵抗尿素和热变性方面的有效性遵循霍夫迈斯特序列：K(+) > Na(+) > Li(+)。
