Sierks M R, Sico C, Zaw M
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County 21250, USA.
Biotechnol Prog. 1997 Sep-Oct;13(5):601-8. doi: 10.1021/bp9700824.
Release of product from the active site is the rate-limiting step in a number of enzymatic reactions, including maltose hydrolysis by glucoamylase (GA). With GA, an enzymatic conformational change has been associated with the product release step. Solvent characteristics such as viscosity can strongly influence protein conformational changes. Here we show that the rate-limiting step of GA has a rather complex dependence on solvent characteristics. Seven different cosolvents were added to the GA/maltose reaction solution. Five of the cosolvents, all having an ethylene glycol base, resulted in an increase in activity at low concentration of cosolvent and variable decreases in activity at higher concentrations. The increase in enzyme activity was dependent on polymer length of the cosolvent; the longer the polymer, the lower the concentration needed. The maximum increase in catalytic activity at 45 degrees C (40-45%) was obtained with the three longest polymers (degree of polymerization from 200 to 8000). A further increase in activity to 60-65% was obtained at 60 degrees C. The linear relationship between ln(kcat) and (viscosity)2 obtained with all the cosolvents provides further evidence that product release is the rate-limiting step in the GA catalytic mechanism. A substantial increase in the turnover rate of GA by addition of relatively small amounts of a cosolvent has potential applications for the food industry where high-fructose corn syrup (HFCS) is one of the primary products produced with GA. Since maltodextrin hydrolysis by GA is by far the slowest step in the production of HFCS, increasing the catalytic rate of GA can substantially reduce the process time.
产物从活性位点释放是许多酶促反应中的限速步骤，包括葡糖淀粉酶（GA）催化的麦芽糖水解反应。对于GA而言，酶的构象变化与产物释放步骤相关。诸如粘度等溶剂特性会强烈影响蛋白质的构象变化。在此我们表明，GA的限速步骤对溶剂特性具有相当复杂的依赖性。向GA/麦芽糖反应溶液中添加了七种不同的共溶剂。其中五种共溶剂均以乙二醇为基础，在低浓度共溶剂时导致活性增加，而在较高浓度时活性则有不同程度的降低。酶活性的增加取决于共溶剂的聚合物长度；聚合物越长，所需浓度越低。使用三种最长的聚合物（聚合度从200到8000）在45℃时催化活性最大增加（40 - 45%）。在60℃时活性进一步增加至60 - 65%。所有共溶剂的ln(kcat)与(粘度)²之间的线性关系进一步证明产物释放是GA催化机制中的限速步骤。通过添加相对少量的共溶剂使GA的周转速率大幅提高，这对于食品工业具有潜在应用价值，其中高果糖玉米糖浆（HFCS）是用GA生产的主要产品之一。由于GA催化的麦芽糊精水解是HFCS生产中迄今为止最慢的步骤，提高GA的催化速率可大幅缩短生产时间。
