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Domain Structure and Function of Gluoamylase

葡萄糖淀粉酶的结构域和功能

基本信息

批准号：
05660089
负责人：
TANAKA Akiyoshi
金额：
$ 1.28万
依托单位：
MIE UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05660089/
关键词：
Glucoamylase Domain Structure Subsite Structure Calorimetry Kinetics Isozyme Random Attack Starch Binding Domain DSC 等温滴定熱量計

项目摘要

Calorimetric and steady-state kinetic studies were done to investigate the structure and function of fungal glucoamylases.(1) Thermal unfolding of two forms of Aspergillus niger glucoamylase was observed by adiabatic differential scanning calorimetry (DSC) at pH 7. The DSC traces of the larger form of the enzyme, G1, and the shorter form which lacks the C-terminal starch-binding domain of G1, G2, could be resolved by assuming two-state unfolding of five and four independent components, respectively. The thermal unfolding of the starch-binding domain was found to be reversible, but that of the catalytic domain was irreversible. The DSC observation of G1 and G2 in the presence of beta-cyclodextrin or 1-deoxynojirimycin showed that there was no domain interaction between the starch-binding domain and catalytic domain.(2) Steady-state inhibitory kinetic studies of Aspergillus glucoamylase showed that gluconolactone and 1-deoxynojirimycin were non-competitive type and competitive type inihi … More bitor, respectively. These results indicate that gluconolactone binds to a site other than the active site of the enzyme forming nonproductive ternary complex of the enzyme, substrate, and gluconolactone, but 1-deoxynojirimycin binds to the active site (most probably subsite 2) of the enzyme without forming the ternary complex.(3) The subsite structure of Rhizopus glucoamylase was identified for the hydrolytic reaction of isomaltooligosaccharides at 25ﾟC and at pH4.5. The arrangement of the subsite affinities A_i's (i is the subsite number) was evaluated as follows : A_1=-6.0, A_2=13.8, A_3=4.6, A_4=1.5, A_5=0.6, A_6=-0.5, and A_7=0.2 (in kj mol^<-1>) unit). The intrinsic rate constant for the hydrolytic reaction in the productive binding mode of substrates, k_<int>, was 10.2 s^<-1>. Larger values of K_m for isomaltooligosaccharides compared with those for maltooligosaccharides were attributed to smaller values A_1 and A_2 compared with those for maltooligosaccharides, and smaller values of k_0 were attributed t smaller values of A_1 and k_<int>.(4) Hydrolytic reactions of maltooligosaccharides (degree of polymerization n=3-7) and isomaltooligosaccharides (n=3-7) by Rhizopus glucoamylase were observed by HPLC at pH 4.5,25ﾟC.It was confirmed that initial rates of the decrease of n-mer substrate and the increase of the products, glucose and (n-1)-mer oligosaccharide, are the same for all the substrates, indicating that the reaction mode of the enzyme is "random attack, " and no "multiple attack" or "single chain attack" is operating. Less

对真菌葡糖淀粉酶的结构和功能进行了量热和稳态动力学研究。(1)采用绝热差示扫描量热法（DSC）研究了尼日尔曲霉葡萄糖淀粉酶在pH 7时的热去折叠行为。的DSC痕迹的较大形式的酶，G1，和较短的形式，缺乏C-末端淀粉结合结构域的G1，G2，可以解决通过假设两个状态展开的五个和四个独立的组件，分别。淀粉结合结构域的热去折叠是可逆的，而催化结构域的热去折叠是不可逆的。G1和G2在β-环糊精或1-脱氧野尻霉素存在下的DSC观察表明，淀粉结合结构域和催化结构域之间没有结构域相互作用。(2)对曲霉葡萄糖淀粉酶的稳态抑制动力学研究表明，曲霉内酯和1-脱氧野尻霉素分别为非竞争型和竞争型抑制动力学 ...更多信息，分别。这些结果表明，野尻内酯与酶的活性位点以外的位点结合，形成酶、底物和野尻内酯的非生产性三元复合物，但1-脱氧野尻霉素与酶的活性位点（最可能是亚位点2）结合，而不形成三元复合物。(3)在25 ℃和pH4.5条件下，根霉葡萄糖淀粉酶水解低聚异麦芽糖的亚位点结构被确定。亚位亲和力A_i（i为亚位数）的排列如下：A_1=-6.0，A_2 =13.8，A_3=4.6，A_4=1.5，A_5=0.6，A_6=-0.5，A_7 =0.2（单位为kj mol·L-<-1>1）。在底物的生产性结合模式中，水解反应的本征速率常数k1<int>为10.2s-1<-1>。低聚异麦芽糖的Km值大于低聚麦芽糖的Km值是由于A_1和A_2值小于低聚麦芽糖的Km值，而k_0值小于低聚麦芽糖的Km值是由于A_1和k_2值小于低聚麦芽糖的Km值<int>。(4)麦芽低聚糖的水解反应在pH4.5，25 ℃条件下，用高效液相色谱法对根霉葡萄糖淀粉酶（n = 3 -7）和异麦芽低聚糖（n=3-7）的反应进行了研究，结果表明，所有底物的n-聚体底物减少和产物葡萄糖和（n-1）-聚体低聚糖增加的初始速率是相同的，表明该酶的反应模式为“随机攻击“，不存在“多重攻击”或“单链攻击”。少