Mapping the extended substrate binding site of cathepsin G and human leukocyte elastase. Studies with peptide substrates related to the alpha 1-protease inhibitor reactive site.
Mapping the extended substrate binding site of cathepsin G and human leukocyte elastase. Studies with peptide substrates related to the alpha 1-protease inhibitor reactive site.
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DOI：
10.1016/s0021-9258(18)50690-6
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发表时间：
1979-05
期刊：
影响因子：
--
通讯作者：
K. Nakajima;J. Powers;B. Ashe;M. Zimmerman
中科院分区：
文献类型：
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作者：
K. Nakajima;J. Powers;B. Ashe;M. Zimmerman
本文报道了人白细胞(HL)弹性酶和组织蛋白酶G、猪胰腺弹性酶和牛胰凝乳蛋白酶在pH 7.50时对一系列I-硝基苯胺底物的水解反应的动力学常数。HL弹性蛋白酶和组织蛋白酶G目前被认为是导致肺气肿肺部破坏的因素。MeO-Sue-Ala-Ala-Pro-Vai-NA是HL弹性酶的优良底物，不被组织蛋白酶G水解。MeO-Sue基团相对于乙酰基增加了底物的溶解度。对于HL弹性酶，这种结构变化增加了该酶对I-硝基苯胺底物和氯甲基酮抑制剂的反应活性。这表明HL弹性蛋白酶与底物(或抑制物)的至少5个残基相互作用。组织蛋白酶G喜欢带负电荷的铅基团，如Sue-、Suc(4F)-、GLT-或Mal-。这种酶和许多其他丝氨酸蛋白酶一样，不能接受其SS亚基上的Pro残基。组织蛋白酶G较好的底物Sue-Ala-Ala-Pro-Phe-NA不被HL弹性酶降解。这些工具对研究HL弹性蛋白酶和组织蛋白酶G的生物学功能是有用的。我们研究了两种与血浆反应部位有关的四肽4-硝基苯胺底物(~1‘蛋白酶抑制剂(ai-抗胰蛋白酶))。两者都有一个P1 Met残基，而一个MeO-Suc-Ala-Ile-Pro-Met-NA在al-PI的蛋白水解点上具有pA到pT残基的准确序列(Johnson，DA和Travis，J.(1978)J.Biol Clrem)。253、7142-7144)。MeO-Sue-Ala-Ala-Pro-Met-NA和MeO-Suc-Ala-Ile-Pro-Met-NA都能与组织蛋白酶G、HL弹性蛋白酶和牛胰凝乳酶反应。前者实际上是迄今报道的组织蛋白酶G最好的4-硝基苯胺底物。MeO-Sue-Ala-Ala-Pro-Met-Na氧化生成两个非对映异构体亚砜。既不与HL弹性蛋白酶结合，也不被HL弹性蛋白酶或组织蛋白酶G所降解，两者与牛胰凝乳酶反应较差。在前一篇论文中，Johnson和Travis(Johnson，D.和Travis，J.(1979)J.Biol Chem.254,4022-4026)表明al-PI的氧化破坏了其抑制活性。总之，我们的结果表明，(Ye-PI)的等电点Met的氧化能够破坏其对大多数丝氨酸蛋白酶的反应性。香烟烟雾中的某些成分使al-Pi被氧化，这将为这项研究提供一种解释--这项研究部分得到了烟草研究委员会的资助。这篇文章的出版费用部分由支付页面费支付。因此，根据18种用法，该物品必须在此注明“广告”。第1734条只为表明这放屁。$可将信件收件人。
The kinetic constants for the hydrolysis of a series of I-nitroanilide substrates by human leukocyte (HL) elastase and cathepsin G, porcine pancreatic elastase, and bovine chymotrypsin at pH 7.50 are reported. HL elastase and cathepsin G are currently thought to be the agents responsible for destruction of the lung in the disease emphysema. MeO-Sue-Ala-Ala-Pro-VaI-NA is an excellent substrate for HL elastase and is not hydrolyzed by cathepsin G. The MeO-Sue-group increases the solubility of a substrate relative to the acetyl group. With HL elastase, this structural change increases the reactivity of the enzyme toward both I-nitroanilide substrates and chloromethyl ketone inhibitors. This indicates that HL elastase is interacting with at least 5 residues of a substrate (or inhibitor). Cathepsin G prefers Pb groups which are negatively charged such as Sue-, Suc (4F)-, Glt-, or Mal-. This enzyme, in common with many other serine proteases cannot accept a Pro residue at its Ss subsite. One of the better substrates for cathepsin G, Sue-Ala-Ala-Pro-Phe-NA, was not hydrolyzed by HL elastase. These tools should be useful in the study of the biological function of HL elastase and cathepsin G. Two tetrapeptide 4-nitroanilide substrates related to the reactive site of the plasma (~ 1’protease inhibitor (ai-antitrypsin) were studied. Both have a P1 Met residue and one, MeO-Suc-Ala-Ile-Pro-Met-NA, has the exact sequence of the Pa to Pt residues at the proteolysis site of al-PI (Johnson, DA, and Travis, J.(1978) J. BioL Clrem. 253, 7142-7144). Both MeO-Sue-Ala-Ala-Pro-Met-NA and MeO-Suc-Ala-Ile-Pro-Met-NA react with cathepsin G, HL elastase, and bovine chymotrypsin. The former is in fact the best 4-nitroanilide substrate of cathepsin G yet reported. Oxidation of MeO-Sue-Ala-Ala-Pro-Met-NA yielded two diastereomeric sulfoxides. Neither are bound to or was hydrolyzed by HL elastase or cathepsin G. Both reacted poorly with bovine chymotrypsin. In the preceding paper, Johnson and Travis (Johnson, D., and Travis, J.(1979) J. BioL Chem. 254, 4022-4026) show that oxidation of al-PI destroys its inhibitory activity. In concert, our results indicate that oxidation of the PI Met of (Ye-PI is capable to destroying its reactivity toward most serine proteases. Oxidation of al-PI by some component in cigarette smoke would offer one explanation in mo-* This investigation was supported in part by a grant from the Council for Tobacco Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USE. Section 1734 solely to indicate this fart. $ To whom correspondence may be addressed.