Vihinen M, Peltonen T, Iitiä A, Suominen I, Mäntsälä P
Department of Biochemistry, University of Turku, Finland.
Protein Eng. 1994 Oct;7(10):1255-9. doi: 10.1093/protein/7.10.1255.
A series of truncated proteins from a thermostable Bacillus stearothermophilus alpha-amylase was prepared to study the importance of the extension in the C-terminus compared with other liquefying Bacillus alpha-amylases. The mutations introducing new translation termination sites shortened the 515 amino acid residue-long wild type enzyme by 17, 32, 47, 73 or 93 residues. The longer the truncation, the lower the specific activity of the enzyme. Only the two longest mutant proteins were active: the specific activity of the 498 residue variant was 97% and protein 483 was 36% that of the parental enzyme. The Km values of starch hydrolysis changed from 1.09 for wild type enzyme to 0.35 and 0.21 for mutants 498 and 483, respectively, indicating altered substrate binding. The mutant enzymes had almost identical pH and temperature optima with the wild type amylase, but enhanced thermal stability and altered end product profile. The consequences of the truncation to the structure and function of the enzymes were explored with molecular modeling. The liquefying amylases seem to require approximately 480 residues to be active, whereas the C-terminal end of B.stearothermophilus amylase is required for increased activity.
制备了一系列来自嗜热脂肪芽孢杆菌的截短热稳定α-淀粉酶蛋白，以研究与其他液化芽孢杆菌α-淀粉酶相比，C末端延伸的重要性。引入新翻译终止位点的突变使515个氨基酸残基长的野生型酶缩短了17、32、47、73或93个残基。截短越长，酶的比活性越低。只有两个最长的突变蛋白具有活性：498个残基变体的比活性为亲本酶的97%，483个残基蛋白的比活性为36%。淀粉水解的Km值从野生型酶的1.09分别变为突变体498和483的0.35和0.21，表明底物结合发生了改变。突变酶与野生型淀粉酶具有几乎相同的最适pH和温度，但热稳定性增强且终产物谱改变。通过分子建模探讨了截短对酶结构和功能的影响。液化淀粉酶似乎需要约480个残基才能具有活性，而嗜热脂肪芽孢杆菌淀粉酶的C末端对于提高活性是必需的。
