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.
Abstract
- 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
- 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。
相似文献
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。
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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。
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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.
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本文引用的文献
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Computer programmes for processing enzyme kinetic data.用于处理酶动力学数据的计算机程序。
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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。
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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。
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