Mattsson P, Battchikova N, Sippola K, Korpela T
Department of Biochemistry, University of Turku, Finland.
Biochim Biophys Acta. 1995 Feb 22;1247(1):97-103. doi: 10.1016/0167-4838(94)00214-2.
Our previous study on cyclomaltodextrin glucanotransferase (CGTase) by chemical modification implied the importance of one or two histidine residues in the cyclization reaction of the enzyme. Based on a computer modelled three-dimensional structure of the CGTase, five histidine residues were chosen as targets for the site-directed mutagenesis. The histidine residues 98, 140, 233 and 327 were replaced by aspartate and His-177 by proline using polymerase chain reaction-mediated techniques. The CGTase variants H98D, H140D, H233D and H327D resulted in a profound decrease in the cyclizing and amylolytic activities, while mutation H177P had little influence on the activities but affected the thermal stability and the width of the pH optimum. It is suggested that His-98 functions as (or as a significant part of) the subsite 2 for the binding of the substrate in CGTase and therefore H98D destabilizes the intermediate for cyclization, but does not markedly affect the hydrolytic reactions. Mutants H140D and H233D produced only minor amounts of alpha-cyclodextrin, did not exhibit substrate inhibition with maltotriose and showed non-Michaelis-Menten kinetics. It is proposed that the variants H140D, H233D and H327D cause steric hindrances near the active center, while mutation H177D has similar consequences on the same site spatially.
我们之前对环糊精葡糖基转移酶（CGTase）进行化学修饰的研究表明，一个或两个组氨酸残基在该酶的环化反应中具有重要作用。基于CGTase的计算机模拟三维结构，选择了五个组氨酸残基作为定点诱变的目标。使用聚合酶链反应介导的技术，将组氨酸残基98、140、233和327替换为天冬氨酸，将His-177替换为脯氨酸。CGTase变体H98D、H140D、H233D和H327D导致环化活性和淀粉分解活性大幅下降，而突变体H177P对活性影响不大，但影响热稳定性和最适pH宽度。有人认为，His-98在CGTase中作为底物结合的亚位点2（或作为其重要组成部分）发挥作用，因此H98D使环化中间体不稳定，但对水解反应没有明显影响。突变体H140D和H233D仅产生少量α-环糊精，对麦芽三糖不表现底物抑制，且呈现非米氏动力学。有人提出，变体H140D、H233D和H327D在活性中心附近造成空间位阻，而突变体H177D在空间上对同一位点有类似影响。
