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1
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.
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 for a two-state transition in papain that may have no close analogue in ficin. Differences in the disposition of cationic sites and hydrophobic binding areas in the active centres of papain and ficin.木瓜蛋白酶中可能在无花果蛋白酶中没有类似情况的双态转变的证据。木瓜蛋白酶和无花果蛋白酶活性中心中阳离子位点和疏水结合区域分布的差异。
Biochem J. 1980 Dec 1;191(3):707-18. doi: 10.1042/bj1910707.
4
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.
5
4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole as a reactivity probe for the investigation of the thiol proteinases. evidence that ficin and bromelain may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain.4-氯-7-硝基苯并-2-恶唑-1,3-二氮唑作为研究硫醇蛋白酶的反应性探针。有证据表明，无花果蛋白酶和菠萝蛋白酶可能缺乏与木瓜蛋白酶天冬氨酸-158构象相当的羧基。
Biochem J. 1976 Nov;159(2):235-44. doi: 10.1042/bj1590235.
6
Characterization of the papain active centre by using two-protonic-state electrophiles as reactivity probes. Evidence for nucleophilic reactivity in the un-interrupted cysteine-25-histidine-159 interactive system.使用双质子态亲电试剂作为反应性探针表征木瓜蛋白酶活性中心。在不间断的半胱氨酸-25-组氨酸-159相互作用系统中亲核反应性的证据。
Biochem J. 1978 May 1;171(2):385-401. doi: 10.1042/bj1710385.
7
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.
8
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.
9
Evidence that binding to the s2-subsite of papain may be coupled with catalytically relevant structural change involving the cysteine-25-histidine-159 diad. Kinetics of the reaction of papain with a two-protonic-state reactivity probe containing a hydrophobic side chain.有证据表明，与木瓜蛋白酶的s2亚位点结合可能与涉及半胱氨酸-25-组氨酸-159二元组的催化相关结构变化相关联。木瓜蛋白酶与含有疏水侧链的双质子态反应性探针反应的动力学。
Biochem J. 1979 Nov 1;183(2):223-31. doi: 10.1042/bj1830223.
10
A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety.一种对硫醇基团具有绝对和选择性特异性的报告基团递送系统以及一种含有7-硝基苯并-2-恶唑-1,3-二氮杂环戊二烯部分的改进型荧光探针。
Biochem J. 1975 Nov;151(2):417-32. doi: 10.1042/bj1510417.

引用本文的文献

1
Isolation and characterization of a protease from the fruit for improving meat tenderness.从水果中分离并鉴定一种用于改善肉质嫩度的蛋白酶。
Food Sci Biotechnol. 2016 Aug 31;25(4):1059-1064. doi: 10.1007/s10068-016-0171-y. eCollection 2016.
2
Human gastrointestinal nematode infections: are new control methods required?人体胃肠道线虫感染：是否需要新的控制方法？
Int J Exp Pathol. 2006 Oct;87(5):325-41. doi: 10.1111/j.1365-2613.2006.00495.x.
3
Temperature-dependences of the kinetics of reactions of papain and actinidin with a series of reactivity probes differing in key molecular recognition features.木瓜蛋白酶和猕猴桃蛋白酶与一系列在关键分子识别特征上不同的反应性探针反应动力学的温度依赖性。
Biochem J. 2006 May 15;396(1):17-21. doi: 10.1042/BJ20051501.
4
Isomerization of the uncomplexed actinidin molecule: kinetic accessibility of additional steps in enzyme catalysis provided by solvent perturbation.未络合的猕猴桃蛋白酶分子的异构化：溶剂扰动提供的酶催化中其他步骤的动力学可达性。
Biochem J. 2004 Mar 1;378(Pt 2):699-703. doi: 10.1042/BJ20031318.
5
Variation in the pH-dependent pre-steady-state and steady-state kinetic characteristics of cysteine-proteinase mechanism: evidence for electrostatic modulation of catalytic-site function by the neighbouring carboxylate anion.半胱氨酸蛋白酶机制中pH依赖性预稳态和稳态动力学特征的变化：相邻羧酸根阴离子对催化位点功能进行静电调节的证据
Biochem J. 2003 Jun 15;372(Pt 3):735-46. doi: 10.1042/BJ20030177.
6
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.
7
Catalytic-site characteristics of the porcine calpain II 80 kDa/18 kDa heterodimer revealed by selective reaction of its essential thiol group with two-hydronic-state time-dependent inhibitors: evidence for a catalytic site Cys/His interactive system and an ionizing modulatory group.通过猪钙蛋白酶II 80 kDa/18 kDa异二聚体的必需巯基与双水合态时间依赖性抑制剂的选择性反应揭示的催化位点特征：催化位点半胱氨酸/组氨酸相互作用系统和电离调节基团的证据。
Biochem J. 1993 Feb 15;290 ( Pt 1)(Pt 1):75-83. doi: 10.1042/bj2900075.
8
The structural origins of the unusual specificities observed in the isolation of chymopapain M and actinidin by covalent chromatography and the lack of inhibition of chymopapain M by cystatin.通过共价色谱法分离木瓜凝乳蛋白酶M和猕猴桃蛋白酶时观察到的异常特异性的结构起源，以及胱抑素对木瓜凝乳蛋白酶M缺乏抑制作用。
Biochem J. 1995 Feb 15;306 ( Pt 1)(Pt 1):39-46. doi: 10.1042/bj3060039.
9
Current problems in mechanistic studies of serine and cysteine proteinases.丝氨酸蛋白酶和半胱氨酸蛋白酶机制研究中的当前问题。
Biochem J. 1982 Oct 1;207(1):1-10. doi: 10.1042/bj2070001.
10
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.

本文引用的文献

1
Computer programmes for processing enzyme kinetic data.用于处理酶动力学数据的计算机程序。
Nature. 1963 May 4;198:463-5. doi: 10.1038/198463a0.
2
Resolution of thiol-containing proteins by sequential-elution covalent chromatography.通过顺序洗脱共价色谱法分离含硫醇的蛋白质。
J Biochem Biophys Methods. 1981 Feb;4(2):101-11. doi: 10.1016/0165-022x(81)90023-3.
3
Evidence for a two-state transition in papain that may have no close analogue in ficin. Differences in the disposition of cationic sites and hydrophobic binding areas in the active centres of papain and ficin.木瓜蛋白酶中可能在无花果蛋白酶中没有类似情况的双态转变的证据。木瓜蛋白酶和无花果蛋白酶活性中心中阳离子位点和疏水结合区域分布的差异。
Biochem J. 1980 Dec 1;191(3):707-18. doi: 10.1042/bj1910707.
4
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.
5
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.
6
The specificity of actinidin and its relationship to the structure of the enzyme.猕猴桃蛋白酶的特异性及其与酶结构的关系。
Biochim Biophys Acta. 1980 Nov 6;616(1):30-4. doi: 10.1016/0005-2744(80)90260-0.
7
Evidence that the lack of high catalytic activity of thiolsubtilisin towards specific substrates may be due to an inappropriately located proton-distribution system. Demonstration of highly nucleophilic character of the thiol group of thiolsubtilisin in the catalytically relevant ionization state of the active centre by use of a two-protonic-state reactivity probe.有证据表明，硫醇枯草杆菌蛋白酶对特定底物缺乏高催化活性可能是由于质子分布系统定位不当。通过使用双质子态反应性探针，证明了在活性中心的催化相关电离状态下硫醇枯草杆菌蛋白酶硫醇基团具有高度亲核性。
Biochem J. 1981 Mar 1;193(3):819-23. doi: 10.1042/bj1930819.
8
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.
9
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.
10
Preliminary crystallographic data for actinidin, a thiol protease from Actinidia chinensis.中华猕猴桃硫醇蛋白酶——肌动蛋白水解酶的初步晶体学数据。
J Mol Biol. 1973 Mar 5;74(3):411-2. doi: 10.1016/0022-2836(73)90382-3.

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。

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.

作者信息

Brocklehurst K, Baines B S, Malthouse J P

出版信息

Biochem J. 1981 Sep 1;197(3):739-46. doi: 10.1042/bj1970739.

DOI:10.1042/bj1970739
PMID:7034724
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1163189/
Abstract
  1. A rapid method of isolation of fully active actinidin, the cysteine proteinase from Actinidia chinensis (Chinese gooseberry or kiwifruit), by covalent chromatography, was devised. 2. The active centre of actinidin was investigated by using n-propyl 2-pyridyl disulphide, 4-(N-aminoethyl 2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole and 4-chloro-7-nitrobenzofurazan as reactivity probes. 3. The presence in actinidin in weakly acidic media of an interactive system containing a nucleophilic sulphur atom was demonstrated. 4. The pKa values (3.1 and 9.6) that characterize this interactive system are more widely separated than those that characterize the interactive active centre systems of ficin (EC 3.4.22.3) and papain (EC 3.4.22.2) (3.8 and 8.6, and 3.9 and 8.8 respectively). 5. Actinidin was shown to resemble ficin rather than papain in (i) the disposition of the active-centre imidazole group with respect to hydrophobic binding areas, and (ii) the inability of the active-centre aspartic acid carboxy group to influence the reactivity of the active-centre thiol group at pH values of about 4. 6. The implications of the results for one-state and two-state mechanisms for cysteine-proteinase catalysis are discussed.
摘要
  1. 设计了一种通过共价色谱快速分离中华猕猴桃（中国醋栗或奇异果）中半胱氨酸蛋白酶——完全活性的猕猴桃蛋白酶的方法。2. 以正丙基2-吡啶基二硫化物、4-（N-氨乙基2'-吡啶基二硫化物）-7-硝基苯并-2-恶唑-1,3-二唑和4-氯-7-硝基苯并呋咱作为反应探针，研究了猕猴桃蛋白酶的活性中心。3. 证明了在弱酸性介质中猕猴桃蛋白酶中存在一个含有亲核硫原子的相互作用体系。4. 表征该相互作用体系的pKa值（3.1和9.6）比表征无花果蛋白酶（EC 3.4.22.3）和木瓜蛋白酶（EC 3.4.22.2）的相互作用活性中心体系的pKa值（分别为3.8和8.6，以及3.9和8.8）的间隔更大。5. 结果表明，猕猴桃蛋白酶在以下方面类似于无花果蛋白酶而非木瓜蛋白酶：（i）活性中心咪唑基团相对于疏水结合区域的位置，以及（ii）在pH值约为4时，活性中心天冬氨酸羧基无法影响活性中心巯基的反应性。6. 讨论了这些结果对半胱氨酸蛋白酶催化的单态和双态机制的意义。