Increasing the hydrolysis constant of the reactive site upon introduction of an engineered Cys14-Cys39 bond into the ovomucoid third domain from silver pheasant
Increasing the hydrolysis constant of the reactive site upon introduction of an engineered Cys14-Cys39 bond into the ovomucoid third domain from silver pheasant
复制标题
将工程化的 Cys14-Cys39 键引入白雉卵类粘蛋白第三结构域后，增加反应位点的水解常数
DOI：
10.1002/psc.1381
复制
发表时间：
2011
期刊：
影响因子：
--
通讯作者：
and Kobayashi Y
中科院分区：
文献类型：
--
作者：
Hemmi H;Kumazaki T;Kojima S;Yoshida T.;Ohkubo;T;Yokosawa H;Miura K;and Kobayashi Y
P14 C/N39 C是来自银雉（OMSVP 3）的卵类粘蛋白第三结构域的二硫键变体，基于OMSVP 3与海鞘胰蛋白酶抑制剂之间的序列同源性，在反应位点附近引入工程化的Cys 14-Cys 39键。该变体表现出较窄的抑制特异性。我们已经研究了将Cys 14 → Cys 39键引入OMSVP 3的柔性N末端环对反应位点肽键水解的热力学以及反应位点完整抑制剂的热稳定性的影响。P14 C/N39 C可被灰色链霉菌蛋白酶B选择性地在OMSVP 3的反应位点切割，形成反应位点修饰的抑制剂。在任何pH条件下，完整的P14 C/N39 C向修饰的P14 C/N39 C的转化速率都比野生型快得多。P14 C/N39 C的非pH依赖性水解常数（Khyd°）估计约为5.5，高于天然OMSVP 3的值1.6。P14 C/N39 C的反应位点修饰形式在化学上比完整形式更稳定。使用完整抑制剂的热变性实验显示，在pH 2.0下解折叠的中点处的温度对于P14 C/N39 C为59 °C，对于野生型为58 °C。除了P14 C/N39 C之外，还没有引入工程化二硫化物导致Khyd °显著增加但对热稳定性没有影响的实例。将位点特异性二硫键引入天然Kazal型抑制剂的柔性N末端环中将有助于进一步表征反应性位点肽键水解的热力学。版权所有© 2011欧洲肽协会和约翰威利父子有限公司。
P14C/N39C is the disulfide variant of the ovomucoid third domain from silver pheasant (OMSVP3) introducing an engineered Cys14Cys39bond near the reactive site on the basis of the sequence homology between OMSVP3 and ascidian trypsin inhibitor. This variant exhibits a narrower inhibitory specificity. We have examined the effects of introducing a Cys14Cys39bond into the flexible N‐terminal loop of OMSVP3 on the thermodynamics of the reactive site peptide bond hydrolysis, as well as the thermal stability of reactive site intact inhibitors. P14C/N39C can be selectively cleaved byStreptomyces griseusprotease B at the reactive site of OMSVP3 to form a reactive site modified inhibitor. The conversion rate of intact to modified P14C/N39C is much faster than that for wild type under any pH condition. The pH‐independent hydrolysis constant (Khyd°) is estimated to be approximately 5.5 for P14C/N39C, which is higher than the value of 1.6 for natural OMSVP3. The reactive site modified form of P14C/N39C is thermodynamically more stable than the intact one. Thermal denaturation experiments using intact inhibitors show that the temperature at the midpoint of unfolding at pH 2.0 is 59 °C for P14C/N39C and 58 °C for wild type. There have been no examples, except P14C/N39C, where introducing an engineered disulfide causes a significant increase inKhyd°, but has no effect on the thermal stability. The site‐specific disulfide introduction into the flexible N‐terminal loop of natural Kazal‐type inhibitors would be useful to further characterize the thermodynamics of the reactive site peptide bond hydrolysis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.