The Deletion of Amino-Terminal Domain in Thermoactinomyces vulgaris R-47 α-Amylases: Effects of domain N on Activity, Specificity, Stability and Dimerization
The Deletion of Amino-Terminal Domain in Thermoactinomyces vulgaris R-47 α-Amylases: Effects of domain N on Activity, Specificity, Stability and Dimerization
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普通嗜热放线菌 R-47 α-淀粉酶氨基末端结构域的缺失：结构域 N 对活性、特异性、稳定性和二聚化的影响
DOI：
10.1271/bbb.65.401
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发表时间：
2001
期刊：
影响因子：
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通讯作者：
Y. Sakano
中科院分区：
文献类型：
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作者：
T. Yokota;T. Tonozuka;S. Kamitori;Y. Sakano
普通高温放线菌R-47 α-淀粉酶TVA I和TVA II具有N结构域，其是家族13酶中的额外结构。为了研究N结构域在TVA中的作用，我们构建了N结构域缺失的TVA-ΔN突变体和TVA II的Y14，16，68 A和Y 41，82，95 A突变体。TVA-ΔN在碱性条件下不稳定，其热稳定性比野生型低10°C。TVA-ΔN对支链淀粉、淀粉、环糊精和寡糖的比活性急剧下降，比野生型小约1，500至10，000倍。Y14，16，68 A和Y 41，82，95 A对所有底物的kcat值均显著降低，对α-CD和麦芽三糖的Km值分别是野生型的25倍和3倍，对淀粉的Km值是野生型的10倍。溶液中的TVA I和TVA-ΔN是单体，而TVA II是均二聚体，通过其分子量计算。这些结果表明，TVA中的N结构域是一个重要的结构，稳定酶，识别和水解的底物，和TVA II的二聚体。
Thermoactinomyces vulgaris R-47 α-amylases, TVA I and TVA II, have a domain N, which is an extra structure in the family 13 enzymes. To investigate the roles of domain N in TVAs, we constructed TVAs-ΔN mutants which are deleted in domain N, and Y14, 16, 68A and Y41, 82, 95A mutants of TVA II. TVAs-ΔN were unstable under alkaline conditions, and their thermal stabilities were 10°C lower than that of wild-types. The specific activities of TVAs-ΔN for pullulan, starch, cyclodextrins, and oligosaccharides were drastically decreased, being about 1,500- to 10,000-fold smaller than those of wild-types. The kcat values of Y14, 16, 68A and Y41, 82, 95A for all tested substrates were markedly decreased, and the Km value of Y14, 16, 68A for α-CD and maltotriose were 25- and 3-fold larger, and that of Y41, 82, 92A for starch was 10-fold larger than that of the wild-type. TVA I and TVAs-ΔN in solution are a monomer, while TVA II is a homo-dimer, calculated by their molecular masses. These results suggest domain N in TVAs is an important structure for stabilization of enzymes, recognition and hydrolysis of substartes, and dimerization of TVA II.
M. Soejima 等人：“Proteas 无色杆菌水解聚-L-赖氨酸”Agric。
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