Chen Y, Wu J W, Xu G J, Tsou C L, Wang Z X
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Bejiing, China.
Eur J Biochem. 1997 Sep 15;248(3):925-9. doi: 10.1111/j.1432-1033.1997.00925.x.
The course of inactivation of the reduced spinach chloroplast fructose-1,6-bisphosphatase by digestion with subtilisin has been followed by the progress curve method [Tsou, C. L. (1988) Adv. Enzymol. 61, 381-436] and found to follow first-order kinetics. On the basis of the hydrolysis of the substrate, fructose 1,6-bisphosphate, at different concentrations during proteolysis by subtilisin, the first-order inactivation rate constants for the free enzyme and the enzyme-substrate complex can both be determined. The ratio between the inactivation rate constants for the free enzyme and the enzyme-substrate complex indicates strong protection against subtilisin proteolysis by the substrate. It is proposed that the above ratio can be used as a quantitative measure of substrate protection for enzyme inactivation generally. As it has been found that the site of proteolysis is located in a loop region near the N-terminus and well away from the active site, the substrate protection indicates a conformation change of the enzyme away from the substrate binding site.
用枯草杆菌蛋白酶消化还原型菠菜叶绿体果糖-1,6-二磷酸酶的失活过程，已通过进程曲线法[邹承鲁（1988年）《酶学进展》61卷，381 - 436页]进行跟踪，发现其遵循一级动力学。基于在枯草杆菌蛋白酶进行蛋白水解期间不同浓度底物果糖1,6 - 二磷酸的水解情况，游离酶和酶 - 底物复合物的一级失活速率常数均可测定。游离酶与酶 - 底物复合物失活速率常数的比值表明底物对枯草杆菌蛋白酶的蛋白水解具有很强的保护作用。有人提出，上述比值一般可作为酶失活的底物保护定量指标。由于已发现蛋白水解位点位于靠近N端的一个环区，且远离活性位点，所以底物保护表明酶发生了远离底物结合位点的构象变化。
