Srivastava R A
Biochemistry Division, Regional Research Laboratory, Jorhat, Assam, India.
Indian J Biochem Biophys. 1991 Apr;28(2):109-13.
With a view to understand the changes in the conformation of bacterial amylase, the enzyme preparation was conjugated to dextran. Glycosylation of purified bacterial amylase resulted in increased stability against heat, proteolytic enzymes and denaturing agents. Several group specific inhibitors exhibited dose-dependent inhibition and the extent of inhibition was same for native as well as for the glycosylated enzyme. The pH optima of native and glycosylated enzyme remained the same indicating that the ionization at the active site is not greatly influenced as a result of glycosylation. Although the native as well as the glycosylated enzyme bind to the substrate with the same affinity, the rate of reaction differed greatly at 90 and 100 degrees C. At 70 degrees C, the rate of reaction was similar for the conjugated as well as the unconjugated amylase. Thermostability at different temperatures clearly showed that the glycosylated enzyme had greater stability compared to the native enzyme. The divalent cation binding site in the amylase also appears to be unaltered upon glycosylation since EDTA inhibited both enzymes to the same extent and addition of calcium ion restored the activity to almost the same level. These studies showed that conjugating the amylase enzyme with a bulky molecule like dextran does not affect the conformation at the active site.
为了了解细菌淀粉酶构象的变化，将酶制剂与葡聚糖偶联。纯化的细菌淀粉酶糖基化导致其对热、蛋白水解酶和变性剂的稳定性增加。几种基团特异性抑制剂表现出剂量依赖性抑制，天然酶和糖基化酶的抑制程度相同。天然酶和糖基化酶的最适pH值保持不变，这表明糖基化并未对活性位点的电离产生很大影响。尽管天然酶和糖基化酶与底物的结合亲和力相同，但在90℃和100℃时反应速率差异很大。在70℃时，偶联淀粉酶和未偶联淀粉酶的反应速率相似。不同温度下的热稳定性清楚地表明，糖基化酶比天然酶具有更高的稳定性。淀粉酶中的二价阳离子结合位点在糖基化后似乎也未改变，因为EDTA对两种酶的抑制程度相同，添加钙离子后活性恢复到几乎相同的水平。这些研究表明，将淀粉酶与像葡聚糖这样的大分子偶联不会影响活性位点的构象。
