摘要
比较酶在变性过程中构象和活力变化 ,发现在活性完全丧失时尚无可察觉的整体构象变化。排除变性剂抑制和寡聚酶解聚等可能性之后 ,提出了酶活性部位柔性假说。随后用多种实验方法直接证实了活性部位的构象变化先于分子整体构象变化 ,并与活性丧失同步。根据催化过程中活性部位构象变化 ,以及限制活性部位构象变化对酶活性的影响 ,提出了酶活性部位柔性为酶充分表现其催化活性所必需的设想。
Comparative studies of conformation and activity changes of a number of enzymes during denaturation have shown that inactivation generally precedes detectable global conformational changes. Kinetically, the inactivation rates of enzymes during denaturation are much faster than the rates of global conformational changes under identical conditions. It is suggested that the conformation of enzyme active sites are held together by weaker forces and consequently more flexible compared to the molecule as a whole. Conformational changes at the active sites were demonstrated directly by fluorescent and spin probes introduced at the active site of creatine kinase, glyceraldehyde 3 phosphate dehydrogenase and ribonuclease A. In addition, the susceptibility of ribonuclease A to proteolysis is markedly increased in dilute GuHCl. By separation and N terminal sequence analysis of the peptide fragments liberated by hydrolysis with trypsin or proteinase K, the cleavage points can be identified to show that without gross unfolding of the RNase molecule as a whole, loosening up of active site conformation has indeed occurred during inactivation in dilute GuHCl. For the role of active site flexibility in enzyme catalysis, it is possible that each intermediate step of the whole cycle of catalysis requires the enzyme molecule to be in a different conformation state. Ac [CM(42]tive site flexibility would therefore be essential for the full expression of enzyme activity. It has recently been demonstrated that conformational change, especially that at the active site, accompanies enzyme catalysis and the activation of a number of enzymes involves the loosening up of the active site structure.
出处
《生理科学进展》
CAS
CSCD
北大核心
2001年第1期7-12,共6页
Progress in Physiological Sciences
基金
国家重大基础研究项目"973"资助
关键词
酶
构象
催化机制
活性部位
柔性
Enzyme catalysis
Flexibility
Active site
Conformational change
