Probing the serpin structural-transition mechanism in ovalbumin mutant R339T by proteolytic-cleavage kinetics of the reactive-centre loop
Probing the serpin structural-transition mechanism in ovalbumin mutant R339T by proteolytic-cleavage kinetics of the reactive-centre loop
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DOI：
10.1042/0264-6021:3630403
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发表时间：
2002-04-15
影响因子：
4.1
通讯作者：
Hirose, M
中科院分区：
文献类型：
--
作者：
Arii, Y;Hirose, M
突变的卵清蛋白（R339T），而不是野生型蛋白，在P1-P1'切割后转化为典型的插入环，热稳定的形式[Yamasaki， Arii， Mikami和Hirose (2002) J. Moll。[j].中国生物医学工程学报，2003,18(2):391 - 391。通过蛋白水解裂解动力学研究了卵清蛋白突变体的环插入机制。插入环的性质阻止了枯草菌素对P1-P1‘预裂R339T突变体的进一步切割，而枯草菌素切割了P1-P1’预裂野生型蛋白的第二个P8-P7环。然而，在对完整的R339T进行枯草菌素蛋白水解后，产生了两个最终产物，对应于P1-P1‘单条和P1-P1’/P8-P7双条裂解，根据蛋白水解条件的不同，它们的比例不同。这是由于发生了两个相互竞争的反应：环插入反应和在P1-P1'切割后的直接中间体中第二个P8-P7位点的蛋白水解裂解。这两种反应的竞争性使我们能够建立一种动力学方法来确定反应的速率常数。在R339T突变体中，环插入的一阶速率常数确定为4.0 × 10(-3)/s。R339T突变体P1-P1直接裂解产物中P8-P7的二级速率常数是野生型的10倍。这种高度可接近的环的性质可能在R339T突变型卵清蛋白的环插入机制中起关键作用。
A mutant ovalbumin (R339T), but not the wild-type protein, is transformed into the canonical loop-inserted, thermostabilized form after the P1-P1' cleavage [Yamasaki, Arii, Mikami and Hirose (2002) J. Moll. Biol. 315, 113-120]. The loop-insertion mechanism in the ovalbumin mutant was investigated by proteolytic-cleavage kinetics. The nature of the inserted loop prevented futher cleavage of the P1-P1' pre-cleaved R339T mutant by subtilisin, which cleaved the second P8-P7 loop site in the P1-P1' pre-cleaved wild-type protein. After subtilisin proteolysis of the intact R339T, however, two final products that corresponded to the single P1-P1' and double P1-P1'/P8-P7 cleavages were generated with variable ratios depending on the proteolysis conditions. This was accounted for by the occurrence of two mutually competitive reactions: the loop-insertion reaction and the proteolytic cleavage of the second P8-P7 site in the immediate intermediate after the P1-P1' cleavage. The competitive nature of the two reactions enabled us to establish a kinetic method to determine the rate constants of the reactions. The first-order rate constant for the loop insertion was determined to be 4.0 x 10(-3)/s in the R339T mutant. The second-order rate constant for the P8-P7 cleavage in the immediate P1-P1' cleavage product for the R339T mutant was > 10 times compared with that for its wild-type counterpart. This highly accessible loop nature may play a crucial role in the loop-insertion mechanism for R339T mutant ovalbumin.