OLIGOMERIC STRUCTURE AND SUBSTRATE-INDUCED INHIBITION OF HUMAN CATHEPSIN-C
OLIGOMERIC STRUCTURE AND SUBSTRATE-INDUCED INHIBITION OF HUMAN CATHEPSIN-C
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DOI：
10.1074/jbc.270.37.21626
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发表时间：
1995-09-15
影响因子：
4.8
通讯作者：
TURK, V
中科院分区：
文献类型：
--
作者：
DOLENC, I;TURK, B;TURK, V
组织蛋白酶C已从人肾中用改进的方法纯化。人组织蛋白酶C被分离为pI接近6.0的纯蛋白。该酶的分子量为200 kDa，由四个相同的亚基组成，每个亚基由三条不同的多肽链组成，其中两条是二硫键结合的。测定了它们的氨基末端氨基酸序列。两条链与其他木瓜蛋白酶样半胱氨酸蛋白酶的重链和轻链表现出明显的相似性，而第三条链对应于酶的前序列，从而表明前区的相当大一部分仍然结合在成熟的酶中。底物水解的动力学基本上偏离标准Michaelis-Menten动力学，表明在较高的底物浓度下底物抑制。这些数据是由一个连续的合作相互作用模型，其中一个酶分子可以绑定到四个底物分子，但只有二元酶-底物复合物是催化活性的解释。在整个pH活性范围内观察到底物抑制。从pH活性曲线可以得出结论，至少有三个pK（a）值为4.2，6.8和7.7的可电离基团参与底物水解。因此，人组织蛋白酶C似乎与不同来源的其他半胱氨酸蛋白酶有质的不同。
Cathepsin C has been purified from human kidney by a modified procedure. Human cathepsin C was isolated as pure protein with a pI close to 6.0. The enzyme was shown to have a molecular mass of 200 kDa and to consist of four identical subunits, each composed of three different polypeptide chains, two of them disulfide-bound. Their NH2-terminal amino acid sequences were determined. Two chains showed pronounced similarity with the heavy and light chains of other papain-like cysteine proteinases, whereas the third one corresponded to the prosequence of the enzyme, thus showing that a substantial part of the proregion remains bound in the mature enzyme. The kinetics of substrate hydrolysis deviated substantially from standard Michaelis-Menten kinetics, demonstrating substrate inhibition at higher substrate concentrations. These data are explained by a sequential cooperative interaction model, where an enzyme molecule can bind up to four substrate molecules but where only the binary enzyme-substrate complex is catalytically active. Substrate inhibition was observed over the whole range of pH activity. From the pH activity profile it can be concluded that at least three ionizable groups with pK(a) values 4.2, 6.8, and 7.7 are involved in substrate hydrolysis. Human cathepsin C thus appears to differ qualitatively from other cysteine proteinases of different origin.