墨西哥利什曼原虫半胱氨酸蛋白酶CPB亚型之间底物特异性的差异是由一些氨基酸变化介导的。

Maria A Juliano, Darren R Brooks, Paul M Selzer, Hector L Pandolfo, Wagner A S Judice, Luiz Juliano, Morten Meldal, Sanya J Sanderson, Jeremy C Mottram, Graham H Coombs
{"title":"墨西哥利什曼原虫半胱氨酸蛋白酶CPB亚型之间底物特异性的差异是由一些氨基酸变化介导的。","authors":"Maria A Juliano,&nbsp;Darren R Brooks,&nbsp;Paul M Selzer,&nbsp;Hector L Pandolfo,&nbsp;Wagner A S Judice,&nbsp;Luiz Juliano,&nbsp;Morten Meldal,&nbsp;Sanya J Sanderson,&nbsp;Jeremy C Mottram,&nbsp;Graham H Coombs","doi":"10.1111/j.1432-1033.2004.04311.x","DOIUrl":null,"url":null,"abstract":"<p><p>The CPB genes of the protozoan parasite Leishmania mexicana encode stage-regulated cathepsin L-like cysteine proteases that are important virulence factors and are in a tandem array of 19 genes. In this study, we have compared the substrate preferences of two CPB isoforms, CPB2.8 and CPB3, and a H84Y mutant of the latter enzyme, to analyse the roles played by the few amino acid differences between the isoenzymes in determining substrate specificity. CPB3 differs from CPB2.8 at just three residues (N60D, D61N and D64S) in the mature domain. The H84Y mutation mimics an additional change present in another isoenzyme, CPB18. The active recombinant CPB isoenzymes and mutant were produced using Escherichia coli and the S1-S3 and S1'-S3' subsite specificities determined using a series of fluorogenic peptide derivatives in which substitutions were made on positions P3 to P3' by natural amino acids. Carboxydipeptidase activities of CPB3 and H84Y were also observed using the peptide Abz-FRAK(Dnp)-OH and some of its analogues. The kinetic parameters of hydrolysis by CPB3, H84Y and CPB2.8 of the synthetic substrates indicates that the specificity of S3 to S3' subsites is influenced greatly by the modifications at amino acids 60, 61, 64 and 84. Particularly noteworthy was the large preference for Pro in the P2' position for the hydrolytic activity of CPB3, which may be relevant to a role in the activation mechanism of the L. mexicana CPBs.</p>","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2004-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1432-1033.2004.04311.x","citationCount":"24","resultStr":"{\"title\":\"Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes.\",\"authors\":\"Maria A Juliano,&nbsp;Darren R Brooks,&nbsp;Paul M Selzer,&nbsp;Hector L Pandolfo,&nbsp;Wagner A S Judice,&nbsp;Luiz Juliano,&nbsp;Morten Meldal,&nbsp;Sanya J Sanderson,&nbsp;Jeremy C Mottram,&nbsp;Graham H Coombs\",\"doi\":\"10.1111/j.1432-1033.2004.04311.x\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The CPB genes of the protozoan parasite Leishmania mexicana encode stage-regulated cathepsin L-like cysteine proteases that are important virulence factors and are in a tandem array of 19 genes. In this study, we have compared the substrate preferences of two CPB isoforms, CPB2.8 and CPB3, and a H84Y mutant of the latter enzyme, to analyse the roles played by the few amino acid differences between the isoenzymes in determining substrate specificity. CPB3 differs from CPB2.8 at just three residues (N60D, D61N and D64S) in the mature domain. The H84Y mutation mimics an additional change present in another isoenzyme, CPB18. The active recombinant CPB isoenzymes and mutant were produced using Escherichia coli and the S1-S3 and S1'-S3' subsite specificities determined using a series of fluorogenic peptide derivatives in which substitutions were made on positions P3 to P3' by natural amino acids. Carboxydipeptidase activities of CPB3 and H84Y were also observed using the peptide Abz-FRAK(Dnp)-OH and some of its analogues. The kinetic parameters of hydrolysis by CPB3, H84Y and CPB2.8 of the synthetic substrates indicates that the specificity of S3 to S3' subsites is influenced greatly by the modifications at amino acids 60, 61, 64 and 84. Particularly noteworthy was the large preference for Pro in the P2' position for the hydrolytic activity of CPB3, which may be relevant to a role in the activation mechanism of the L. mexicana CPBs.</p>\",\"PeriodicalId\":11817,\"journal\":{\"name\":\"European journal of biochemistry\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2004-09-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1111/j.1432-1033.2004.04311.x\",\"citationCount\":\"24\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"European journal of biochemistry\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1111/j.1432-1033.2004.04311.x\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"European journal of biochemistry","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1111/j.1432-1033.2004.04311.x","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 24

摘要

墨西哥利什曼原虫的CPB基因编码阶段调控的组织蛋白酶l样半胱氨酸蛋白酶,该蛋白酶是重要的毒力因子,由19个基因串联而成。在本研究中,我们比较了CPB两种同工酶CPB2.8和CPB3以及后者的H84Y突变体对底物的偏好,以分析这两种同工酶之间的氨基酸差异在决定底物特异性方面所起的作用。CPB3与CPB2.8在成熟结构域只有三个残基(N60D, D61N和D64S)不同。H84Y突变模拟了另一种同工酶CPB18中存在的额外变化。利用大肠杆菌制备活性重组CPB同工酶和突变体,并利用一系列荧光肽衍生物测定S1-S3和S1'-S3'亚位点特异性,其中P3至P3'位置被天然氨基酸取代。利用肽Abz-FRAK(Dnp)-OH及其类似物观察了CPB3和H84Y的羧基二肽酶活性。CPB3、H84Y和CPB2.8对合成底物的水解动力学参数表明,在60、61、64和84氨基酸上的修饰对S3到S3'亚位的特异性有很大影响。特别值得注意的是,CPB3的水解活性在P2'位置上更倾向于Pro,这可能与L. mexicana cpb的激活机制有关。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes.

The CPB genes of the protozoan parasite Leishmania mexicana encode stage-regulated cathepsin L-like cysteine proteases that are important virulence factors and are in a tandem array of 19 genes. In this study, we have compared the substrate preferences of two CPB isoforms, CPB2.8 and CPB3, and a H84Y mutant of the latter enzyme, to analyse the roles played by the few amino acid differences between the isoenzymes in determining substrate specificity. CPB3 differs from CPB2.8 at just three residues (N60D, D61N and D64S) in the mature domain. The H84Y mutation mimics an additional change present in another isoenzyme, CPB18. The active recombinant CPB isoenzymes and mutant were produced using Escherichia coli and the S1-S3 and S1'-S3' subsite specificities determined using a series of fluorogenic peptide derivatives in which substitutions were made on positions P3 to P3' by natural amino acids. Carboxydipeptidase activities of CPB3 and H84Y were also observed using the peptide Abz-FRAK(Dnp)-OH and some of its analogues. The kinetic parameters of hydrolysis by CPB3, H84Y and CPB2.8 of the synthetic substrates indicates that the specificity of S3 to S3' subsites is influenced greatly by the modifications at amino acids 60, 61, 64 and 84. Particularly noteworthy was the large preference for Pro in the P2' position for the hydrolytic activity of CPB3, which may be relevant to a role in the activation mechanism of the L. mexicana CPBs.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
自引率
0.00%
发文量
0
期刊最新文献
The citation of bibliographic references in biochemical journals. Recommendations 1971. Carbamoylphosphate synthetase from Pseudomonas aeruginosa. Subunit composition, kinetic analysis and regulation. Nuclear magnetic resonance of protamines. A 13C relaxation study of the three main fractions of clupeine. Stereochemistry of the hydrolysis of trehalose by the enzyme trehalase prepared from the flesh fly Sarcophaga barbata. Studies on energy supply for genetic processes. Requirement for membrane potential in Escherichia coli infection by phage T4.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1