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通过中性和阴离子反应性探针揭示的半胱氨酸蛋白酶活性中心性质的显著分级。肌动蛋白水解酶、无花果蛋白酶、木瓜蛋白酶和木瓜蛋白酶A的巯基对4,4'-二吡啶二硫化物和5,5'-二硫代双-(2-硝基苯甲酸)二阴离子的反应特性。

A marked gradation in active-centre properties in the cysteine proteinases revealed by neutral and anionic reactivity probes. Reactivity characteristics of the thiol groups of actinidin, ficin, papain and papaya peptidase A towards 4,4'-dipyridyl disulphide and 5,5'-dithiobis-(2-nitrobenzoate) dianion.

作者信息

Brocklehurst K, Mushiri S M, Patel G, Willenbrock F

出版信息

Biochem J. 1983 Mar 1;209(3):873-9. doi: 10.1042/bj2090873.

DOI:10.1042/bj2090873
PMID:6347181
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1154168/
Abstract
  1. The kinetics of the reactions of the catalytic-site thiol groups of actinidin (the cysteine proteinase from Actinidia chinensis), ficin (EC 3.4.22.3), papain (EC 3.4.22.2) and papaya peptidase A (the other monothiol cysteine proteinase component of Carica papaya) with 4,4'-dipyridyl disulphide (4-Py-S-S-4-Py) and with 5,5'-dithiobis-(2-nitrobenzoate) dianion (Nbs22-) were studied in the pH range approx. 6-10. These studies provided the pH-independent second-order rate constants (k) for the reactions of the two probe reagents with the catalytic-site thiolate anions each in the environment of a neutral histidine side chain where an active-centre carboxy group would be ionized. 2. The ratio R equal to kNbs22-/k4-Py-S-S-4-Py provides an index of the catalytic-site solvation properties of the four cysteine proteinases and varies markedly from one enzyme to another, being 0.80 for papaya peptidase A (0.86 for the model thiol, 2-mercaptoethanol), 29 for actinidin, 0.18 for ficin and 0.015 for papain. These differences appear to derive mainly from the response of the enzyme to the negative charge on Nbs22-. 3. Possible implications of these results for (a) mechanisms of cysteine proteinase catalysis and (b) the possibility of using series of functionally related enzymes in the study of mechanism are discussed.
摘要
  1. 研究了中华猕猴桃中的肌动蛋白酶（一种半胱氨酸蛋白酶）、无花果蛋白酶（EC 3.4.22.3）、木瓜蛋白酶（EC 3.4.22.2）和番木瓜蛋白酶A（番木瓜中另一种单硫醇半胱氨酸蛋白酶成分）的催化位点硫醇基团与4,4'-二吡啶二硫化物（4-Py-S-S-4-Py）以及与5,5'-二硫代双（2-硝基苯甲酸）二价阴离子（Nbs22-）在pH约为6至10范围内的反应动力学。这些研究给出了两种探针试剂与催化位点硫醇阴离子在中性组氨酸侧链环境（活性中心羧基会发生电离）中反应的与pH无关的二级速率常数（k）。2. 比率R等于kNbs22-/k4-Py-S-S-4-Py，它提供了这四种半胱氨酸蛋白酶催化位点溶剂化性质的一个指标，并且在不同酶之间有显著差异，对于番木瓜蛋白酶A为0.80（对于模型硫醇2-巯基乙醇为0.86），对于肌动蛋白酶为29，对于无花果蛋白酶为0.18，对于木瓜蛋白酶为0.015。这些差异似乎主要源于酶对Nbs22-上负电荷的响应。3. 讨论了这些结果对于（a）半胱氨酸蛋白酶催化机制以及（b）在机制研究中使用一系列功能相关酶的可能性的潜在影响。

相似文献

1
A marked gradation in active-centre properties in the cysteine proteinases revealed by neutral and anionic reactivity probes. Reactivity characteristics of the thiol groups of actinidin, ficin, papain and papaya peptidase A towards 4,4'-dipyridyl disulphide and 5,5'-dithiobis-(2-nitrobenzoate) dianion.通过中性和阴离子反应性探针揭示的半胱氨酸蛋白酶活性中心性质的显著分级。肌动蛋白水解酶、无花果蛋白酶、木瓜蛋白酶和木瓜蛋白酶A的巯基对4,4'-二吡啶二硫化物和5,5'-二硫代双-(2-硝基苯甲酸)二阴离子的反应特性。
Biochem J. 1983 Mar 1;209(3):873-9. doi: 10.1042/bj2090873.
2
Characterization of papaya peptidase A as a cysteine proteinase of Carica papaya L. with active-centre properties that differ from those of papain by using 2,2'-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as reactivity probes. Use of two-protonic-state electrophiles in the identification of catalytic-site thiol groups.以2,2'-二吡啶二硫化物和4-氯-7-硝基苯并呋喃为反应探针，表征番木瓜蛋白酶A为番木瓜（Carica papaya L.）的一种半胱氨酸蛋白酶，其活性中心特性与木瓜蛋白酶不同。使用双质子态亲电试剂鉴定催化位点的巯基。
Biochem J. 1982 Jul 1;205(1):205-11. doi: 10.1042/bj2050205.
3
Evidence that the active centre of chymopapain A is different from the active centres of some other cysteine proteinases and that the Brønsted coefficient (beta nuc.) for the reactions of thiolate anions with 2,2'-dipyridyl disulphide may be decreased by reagent protonation.有证据表明，木瓜凝乳蛋白酶A的活性中心与其他一些半胱氨酸蛋白酶的活性中心不同，并且硫醇盐阴离子与2,2'-二吡啶二硫化物反应的布仑斯惕系数（β nuc.）可能会因试剂质子化而降低。
Biochem J. 1980 Jul 1;189(1):189-29. doi: 10.1042/bj1890189.
4
Reactivities of neutral and cationic forms of 2,2'-dipyridyl disulphide towards thiolate anions. Detection of differences between the active centres of actinidin, papain and ficin by a three-protonic-state reactivity probe.2,2'-二吡啶二硫化物的中性和阳离子形式对硫醇盐阴离子的反应活性。通过三质子态反应探针检测猕猴桃蛋白酶、木瓜蛋白酶和无花果蛋白酶活性中心之间的差异。
Biochem J. 1979 Nov 1;183(2):233-8. doi: 10.1042/bj1830233.
5
Chymopapain A. Purification and investigation by covalent chromatography and characterization by two-protonic-state reactivity-probe kinetics, steady-state kinetics and resonance Raman spectroscopy of some dithioacyl derivatives.木瓜凝乳蛋白酶A。通过共价色谱法进行纯化及研究，并利用双质子态反应探针动力学、稳态动力学以及某些二硫代酰基衍生物的共振拉曼光谱进行表征。
Biochem J. 1986 Jan 1;233(1):119-29. doi: 10.1042/bj2330119.
6
Structure-function relationships in the cysteine proteinases actinidin, papain and papaya proteinase omega. Three-dimensional structure of papaya proteinase omega deduced by knowledge-based modelling and active-centre characteristics determined by two-hydronic-state reactivity probe kinetics and kinetics of catalysis.半胱氨酸蛋白酶肌动蛋白水解酶、木瓜蛋白酶和木瓜蛋白酶ω的结构-功能关系。通过基于知识的建模推导木瓜蛋白酶ω的三维结构，以及通过双水合态反应探针动力学和催化动力学确定其活性中心特征。
Biochem J. 1991 Nov 15;280 ( Pt 1)(Pt 1):79-92. doi: 10.1042/bj2800079.
7
Structure of chymopapain M the late-eluted chymopapain deduced by comparative modelling techniques and active-centre characteristics determined by pH-dependent kinetics of catalysis and reactions with time-dependent inhibitors: the Cys-25/His-159 ion-pair is insufficient for catalytic competence in both chymopapain M and papain.糜蛋白酶M的结构：通过比较建模技术推导得出的晚期洗脱糜蛋白酶，以及通过pH依赖性催化动力学和与时间依赖性抑制剂反应确定的活性中心特征：半胱氨酸-25/组氨酸-159离子对对于糜蛋白酶M和木瓜蛋白酶的催化活性而言均不充分。
Biochem J. 1994 Jun 15;300 ( Pt 3)(Pt 3):805-20. doi: 10.1042/bj3000805.
8
A kinetic method for the study of solvent environments of thiol groups in proteins involving the use of a pair of isomeric reactivity probes and a differential solvent effect. Investigation of the active centre of ficin by using 2,2'- and 4,4'- dipyridyl disulphides as reactivity probes.一种用于研究蛋白质中硫醇基团溶剂环境的动力学方法，该方法涉及使用一对异构反应探针和微分溶剂效应。用2,2'-和4,4'-二吡啶二硫化物作为反应探针研究无花果蛋白酶的活性中心。
Biochem J. 1980 Jan 1;185(1):217-22. doi: 10.1042/bj1850217.
9
Reactions of papain and of low-molecular-weight thiols with some aromatic disulphides. 2,2'-Dipyridyl disulphide as a convenient active-site titrant for papain even in the presence of other thiols.木瓜蛋白酶及低分子量硫醇与某些芳香族二硫化物的反应。2,2'-二吡啶基二硫化物作为木瓜蛋白酶便捷的活性位点滴定剂，即使在存在其他硫醇的情况下也适用。
Biochem J. 1973 May;133(1):67-80. doi: 10.1042/bj1330067.
10
Preparation of fully active ficin from Ficus glabrata by covalent chromatography and characterization of its active centre by using 2,2'-depyridyl disulphide as a reactivity probe.通过共价色谱法从光滑榕中制备完全活性的无花果蛋白酶，并使用2,2'-二吡啶基二硫化物作为反应性探针表征其活性中心。
Biochem J. 1976 Nov;159(2):221-34. doi: 10.1042/bj1590221.

引用本文的文献

1
Variation in aspects of cysteine proteinase catalytic mechanism deduced by spectroscopic observation of dithioester intermediates, kinetic analysis and molecular dynamics simulations.通过二硫酯中间体的光谱观察、动力学分析和分子动力学模拟推导的半胱氨酸蛋白酶催化机制各方面的变化。
Biochem J. 2001 Jul 15;357(Pt 2):343-52. doi: 10.1042/0264-6021:3570343.
2
Natural structural variation in enzymes as a tool in the study of mechanism exemplified by a comparison of the catalytic-site structure and characteristics of cathepsin B and papain. pH-dependent kinetics of the reactions of cathepsin B from bovine spleen and from rat liver with a thiol-specific two-protonic-state probe (2,2'-dipyridyl disulphide) and with a specific synthetic substrate (N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide).酶的天然结构变异作为研究机制的工具：以组织蛋白酶B和木瓜蛋白酶催化位点结构及特性的比较为例。牛脾脏和大鼠肝脏组织蛋白酶B与硫醇特异性双质子态探针（2,2'-二吡啶二硫化物）以及特异性合成底物（N-α-苄氧羰基-L-精氨酰-L-精氨酸2-萘酰胺）反应的pH依赖性动力学。
Biochem J. 1984 Sep 15;222(3):805-14. doi: 10.1042/bj2220805.
3
Differences between the electric fields of the catalytic sites of papain and actinidin detected by using the thiol-located nitrobenzofurazan label as a spectroscopic reporter group.使用巯基定位的硝基苯并呋喃标记作为光谱报告基团检测木瓜蛋白酶和猕猴桃蛋白酶催化位点电场之间的差异。
Biochem J. 1984 Jun 1;220(2):609-12. doi: 10.1042/bj2200609.
4
Subsite differences between the active centres of papaya peptidase A and papain as revealed by affinity chromatography. Purification of papaya peptidase A by ionic-strength-dependent affinity adsorption on an immobilized peptide inhibitor of papain.通过亲和色谱揭示木瓜蛋白酶A和木瓜蛋白酶活性中心的亚位点差异。利用木瓜蛋白酶固定化肽抑制剂上离子强度依赖性亲和吸附法纯化木瓜蛋白酶A。
Biochem J. 1984 May 1;219(3):727-33. doi: 10.1042/bj2190727.
5
Investigation of the catalytic site of actinidin by using benzofuroxan as a reactivity probe with selectivity for the thiolate-imidazolium ion-pair systems of cysteine proteinases. Evidence that the reaction of the ion-pair of actinidin (pKI 3.0, pKII 9.6) is modulated by the state of ionization of a group associated with a molecular pKa of 5.5.以苯并呋咱为反应性探针研究猕猴桃蛋白酶的催化位点，该探针可选择性作用于半胱氨酸蛋白酶的硫醇盐-咪唑鎓离子对体系。有证据表明，猕猴桃蛋白酶（pKI 3.0，pKII 9.6）离子对的反应受一个与分子pKa为5.5的基团的电离状态调节。
Biochem J. 1983 Sep 1;213(3):713-8. doi: 10.1042/bj2130713.
6
Preparation of cathepsins B and H by covalent chromatography and characterization of their catalytic sites by reaction with a thiol-specific two-protonic-state reactivity probe. Kinetic study of cathepsins B and H extending into alkaline media and a rapid spectroscopic titration of cathepsin H at pH 3-4.通过共价色谱法制备组织蛋白酶B和H，并通过与硫醇特异性双质子态反应性探针反应来表征其催化位点。对组织蛋白酶B和H在碱性介质中的动力学研究以及在pH 3-4条件下对组织蛋白酶H的快速光谱滴定。
Biochem J. 1985 Apr 15;227(2):511-9. doi: 10.1042/bj2270511.
7
A general framework of cysteine-proteinase mechanism deduced from studies on enzymes with structurally different analogous catalytic-site residues Asp-158 and -161 (papain and actinidin), Gly-196 (cathepsin B) and Asn-165 (cathepsin H). Kinetic studies up to pH 8 of the hydrolysis of N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide catalysed by cathepsin B and of L-arginine 2-naphthylamide catalysed by cathepsin H.通过对具有结构不同的类似催化位点残基（天冬氨酸-158和-161，木瓜蛋白酶和猕猴桃蛋白酶）、甘氨酸-196（组织蛋白酶B）和天冬酰胺-165（组织蛋白酶H）的酶的研究推导得出的半胱氨酸蛋白酶机制的一般框架。对组织蛋白酶B催化的N-α-苄氧羰基-L-精氨酰-L-精氨酸2-萘酰胺水解以及组织蛋白酶H催化的L-精氨酸2-萘酰胺水解在pH 8以下进行的动力学研究。
Biochem J. 1985 Apr 15;227(2):521-8. doi: 10.1042/bj2270521.
8
Chymopapain A. Purification and investigation by covalent chromatography and characterization by two-protonic-state reactivity-probe kinetics, steady-state kinetics and resonance Raman spectroscopy of some dithioacyl derivatives.木瓜凝乳蛋白酶A。通过共价色谱法进行纯化及研究，并利用双质子态反应探针动力学、稳态动力学以及某些二硫代酰基衍生物的共振拉曼光谱进行表征。
Biochem J. 1986 Jan 1;233(1):119-29. doi: 10.1042/bj2330119.
9
The electrostatic fields in the active-site clefts of actinidin and papain.猕猴桃蛋白酶和木瓜蛋白酶活性位点裂隙中的静电场。
Biochem J. 1988 Aug 15;254(1):235-8. doi: 10.1042/bj2540235.
10
Differences in the chemical and catalytic characteristics of two crystallographically 'identical' enzyme catalytic sites. Characterization of actinidin and papain by a combination of pH-dependent substrate catalysis kinetics and reactivity probe studies targeted on the catalytic-site thiol group and its immediate microenvironment.两个晶体学上“相同”的酶催化位点在化学和催化特性上的差异。通过结合pH依赖性底物催化动力学以及针对催化位点硫醇基团及其紧邻微环境的反应性探针研究，对猕猴桃蛋白酶和木瓜蛋白酶进行表征。
Biochem J. 1987 Oct 1;247(1):181-93. doi: 10.1042/bj2470181.

本文引用的文献

1
A colorimetric method for determining low concentrations of mercaptans.一种测定低浓度硫醇的比色法。
Arch Biochem Biophys. 1958 Apr;74(2):443-50. doi: 10.1016/0003-9861(58)90014-6.
2
Evidence for a close similarity in the catalytic sites of papain and ficin in near-neutral media despite differences in acidic and alkaline media. Kinetics of the reactions of papain and ficin with chloroacetate.尽管在酸性和碱性介质中存在差异，但木瓜蛋白酶和无花果蛋白酶在近中性介质中的催化位点具有高度相似性的证据。木瓜蛋白酶和无花果蛋白酶与氯乙酸反应的动力学。
Biochem J. 1982 Jan 1;201(1):101-4. doi: 10.1042/bj2010101.
3
Differences in the interaction of the catalytic groups of the active centres of actinidin and papain. Rapid purification of fully active actinidin by covalent chromatography and characterization of its active centre by use of two-protonic-state reactivity probes.猕猴桃蛋白酶和木瓜蛋白酶活性中心催化基团相互作用的差异。通过共价色谱法快速纯化完全活性的猕猴桃蛋白酶，并使用双质子态反应性探针表征其活性中心。
Biochem J. 1981 Sep 1;197(3):739-46. doi: 10.1042/bj1970739.
4
Structure of actinidin, after refinement at 1.7 A resolution.经1.7埃分辨率精修后的猕猴桃蛋白酶结构。
J Mol Biol. 1980 Aug 25;141(4):441-84. doi: 10.1016/0022-2836(80)90255-7.
5
A kinetic method for the study of solvent environments of thiol groups in proteins involving the use of a pair of isomeric reactivity probes and a differential solvent effect. Investigation of the active centre of ficin by using 2,2'- and 4,4'- dipyridyl disulphides as reactivity probes.一种用于研究蛋白质中硫醇基团溶剂环境的动力学方法，该方法涉及使用一对异构反应探针和微分溶剂效应。用2,2'-和4,4'-二吡啶二硫化物作为反应探针研究无花果蛋白酶的活性中心。
Biochem J. 1980 Jan 1;185(1):217-22. doi: 10.1042/bj1850217.
6
Characterization of papaya peptidase A as a cysteine proteinase of Carica papaya L. with active-centre properties that differ from those of papain by using 2,2'-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as reactivity probes. Use of two-protonic-state electrophiles in the identification of catalytic-site thiol groups.以2,2'-二吡啶二硫化物和4-氯-7-硝基苯并呋喃为反应探针，表征番木瓜蛋白酶A为番木瓜（Carica papaya L.）的一种半胱氨酸蛋白酶，其活性中心特性与木瓜蛋白酶不同。使用双质子态亲电试剂鉴定催化位点的巯基。
Biochem J. 1982 Jul 1;205(1):205-11. doi: 10.1042/bj2050205.
7
Two-protonic-state electrophiles as probes of enzyme mechanisms.双质子态亲电试剂作为酶作用机制的探针
Methods Enzymol. 1982;87:427-69. doi: 10.1016/s0076-6879(82)87026-2.
8
Determination of sulfhydryl groups with 2,2'- or 4,4'-dithiodipyridine.用2,2'-或4,4'-二硫代二吡啶测定巯基
Arch Biochem Biophys. 1967 Mar;119(1):41-9. doi: 10.1016/0003-9861(67)90426-2.
9
Anionic proteinase from Actinidia chinensis. Preparation and properties of the crystalline enzyme.中华猕猴桃阴离子蛋白酶。结晶酶的制备及性质
Eur J Biochem. 1970 Jun;14(2):214-21. doi: 10.1111/j.1432-1033.1970.tb00280.x.
10
Covalent chromatography. Preparation of fully active papain from dried papaya latex.共价色谱法。从干燥的木瓜乳胶中制备全活性木瓜蛋白酶。
Biochem J. 1973 Jul;133(3):573-84. doi: 10.1042/bj1330573.