Conformational instability of the N‐ and C‐terminal lobes of porcine pepsin in neutral and alkaline solutions
Conformational instability of the N‐ and C‐terminal lobes of porcine pepsin in neutral and alkaline solutions
复制标题
中性和碱性溶液中猪胃蛋白酶 N 和 C 末端叶的构象不稳定性
作者：
Xinli Lin;J. Loy;F. Sussman;Jordan Tang
胃蛋白酶在单链中含有两个构象同源的叶，这两个叶被认为是通过基因复制和融合进化而来的。我们已经证明，单个重组叶能够独立折叠和重构成双链胃蛋白酶或双链胃蛋白酶原（Lin，X.，例如，1992，J.Biol.Chem.267，17257-17263）。胃蛋白酶在中性或碱性溶液中自发失活。我们在本研究中表明，碱性失活胃蛋白酶的酶活性通过添加重组N端叶而不是C端叶再生。这些结果表明，胃蛋白酶的碱性失活是由于其N端叶的选择性变性。已经分离出胃蛋白酶原的重组N末端叶和碱变性胃蛋白酶之间的复合物。这种复合物在结构上类似于双链胃蛋白酶原，但它含有变性胃蛋白酶N末端叶的延伸。复合物的酸化伴随着前区的切割和变性N端叶的蛋白水解。导致N端叶碱性不稳定的结构组分可能是pKa值异常高的羧基。基于结构域三级结构中相互作用电荷的能量学，计算了胃蛋白酶N端结构域中23个净羧基的静电势（与C端结构域中的19个相比）。最可能引起碱性变性的基团是Asp 11、Asp 159、Glu 4、Glu 13和Asp 118。特别是，部分掩埋的Asp 11与Asp 159相互作用，可能导致这两个基团中的一个具有异常高的pKa值，另一个具有异常低的pKa值。因此，Asp 11在高pH下的电离可能会使两个带负电荷的残基非常接近。这种不利的情况可能是胃蛋白酶N端叶变性的触发因素。
Pepsin contains, in a single chain, two conformationally homologous lobes that are thought to have been evolutionarily derived by gene duplication and fusion. We have demonstrated that the individual recombinant lobes are capable of independent folding and reconstitution into a two‐chain pepsin or a two‐chain pepsinogen (Lin, X., et al., 1992, J. Biol. Chem. 267, 17257–17263). Pepsin spontaneously inactivates in neutral or alkaline solutions. We have shown in this study that the enzymic activity of the alkaline‐inactivated pepsin was regenerated by the addition of the recombinant N‐terminal lobe but not by the C‐terminal lobe. These results indicate that alkaline inactivation of pepsin is due to a selective denaturation of its N‐terminal lobe. A complex between recombinant N‐terminal lobe of pepsinogen and alkaline‐denatured pepsin has been isolated. This complex is structurally similar to a two‐chain pepsinogen, but it contains an extension of a denatured pepsin N‐terminal lobe. Acidification of the complex is accompanied by a cleavage in the pro region and proteolysis of the denatured N‐terminal lobe. The structural components that are responsible for the alkaline instability of the N‐terminal lobe are likely to be carboxyl groups with abnormally high pKa values. The electrostatic potentials of 23 net carboxyl groups in the N‐terminal domain (as compared to 19 in the C‐terminal domain) of pepsin were calculated based on the energetics of interacting charges in the tertiary structure of the domain. The groups most probably causing the alkaline denaturation are Asp11, Asp159, Glu4, Glu13, and Asp118. Especially, the partially buried Asp11, which interacts with Asp159, could cause one of these two groups to have an abnormally high pKa and the other an abnormally low pKa value. Thus, the ionization of Asp11 at a high pH may place two negatively charged residues in close vicinity. This unfavorable situation may be the trigger for the denaturation of the N‐terminal lobe of pepsin.
胃蛋白酶、根霉胃蛋白酶及其活性位点氢键突变体动力学参数的 pH 依赖性。
DOI：
--
发表时间：
1992
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Lin,Y;Fusek,M;Lin,X;Hartsuck,JA;Kezdy,FJ;Tang,J
通讯作者：
Tang,J
双链胃蛋白酶原、双链胃蛋白酶和胃蛋白酶原氨基末端叶的酶活性。
DOI：
--
发表时间：
1992
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Lin,XL;Lin,YZ;Koelsch,G;Gustchina,A;Wlodawer,A;Tang,J
通讯作者：
Tang,J
影响因子：
56.9
作者：
WLODAWER, A;MILLER, M;KENT, SBH
通讯作者：
KENT, SBH