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。
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.
作者信息
Brocklehurst K, Malthouse J P
出版信息
Biochem J. 1980 Dec 1;191(3):707-18. doi: 10.1042/bj1910707.
Abstract
The kinetics of the reactions of the active-centre thiol groups of papain (EC 3.4.22.2) and ficin (EC 3.4.22.3) with the two-protonic-state reactivity probes 2,2'-dipyridyl disulphide, n-propyl 2-pyridyl disulphide and 4-(N-aminoethyl 2'-pyridyl disulphide)- 7-nitrobenzo-2-oxa-1,3-diazole (compound I) were studied over a wide range of pH. Differences between the reactivities of ficin and papain towards the cationic forms of the alkyl 2-pyridyl disulphide probes suggest that ficin contains a cationic site without exact analogue in papain, and the striking difference in the shapes of the pH-rate profiles for the reactions of the two enzymes with compound (1) suggests differences in the mobilities or dispositions of the active-centre histidine imidazole groups with respect to relevant hydrophobic binding areas. The evidence from reactivity-probe studies that the papain catalytic mechanism involves substantial repositioning of the active-centre imidazole group during the catalytic act does not apply also to ficin. If ficin contains an aspartic acid residue analogous to aspartic acid-158 in papain, the pKa of its carboxy group is probably significantly lower than the pKa of the analogous group in papain.
摘要
在较宽的pH范围内，研究了木瓜蛋白酶（EC 3.4.22.2）和无花果蛋白酶（EC 3.4.22.3）活性中心巯基与双质子态反应性探针2,2'-二吡啶二硫化物、正丙基2-吡啶二硫化物和4-(N-氨乙基2'-吡啶二硫化物)-7-硝基苯并-2-恶唑-1,3-二唑（化合物I）的反应动力学。无花果蛋白酶和木瓜蛋白酶对烷基2-吡啶二硫化物探针阳离子形式的反应性差异表明，无花果蛋白酶含有一个在木瓜蛋白酶中没有确切类似物的阳离子位点，并且两种酶与化合物（1）反应的pH-速率曲线形状的显著差异表明活性中心组氨酸咪唑基团相对于相关疏水结合区域的迁移率或位置存在差异。反应性探针研究表明木瓜蛋白酶催化机制在催化过程中涉及活性中心咪唑基团的大量重新定位，这一证据并不适用于无花果蛋白酶。如果无花果蛋白酶含有一个与木瓜蛋白酶中天冬氨酸-158类似的天冬氨酸残基，其羧基的pKa可能明显低于木瓜蛋白酶中类似基团的pKa。
相似文献
1
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.
2
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.
3
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.
4
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.
5
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
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。
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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。
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9
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.
10
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。
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引用本文的文献
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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.
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.
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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。
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Biochem J. 1982 Oct 1;207(1):1-10. doi: 10.1042/bj2070001.
5
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。
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7
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.
8
Effects of conformational selectivity and of overlapping kinetically influential ionizations on the characteristics of pH-dependent enzyme kinetics. Implications of free-enzyme pKa variability in reactions of papain for its catalytic mechanism.构象选择性和重叠动力学影响电离对pH依赖性酶动力学特征的影响。木瓜蛋白酶反应中游离酶pKa变异性对其催化机制的影响。
Biochem J. 1983 Jun 1;211(3):701-8. doi: 10.1042/bj2110701.
9
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.
本文引用的文献
1
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.
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On the size of the active site in proteases. I. Papain.关于蛋白酶活性位点的大小。I. 木瓜蛋白酶。
Biochem Biophys Res Commun. 1967 Apr 20;27(2):157-62. doi: 10.1016/s0006-291x(67)80055-x.
3
On the active site of proteases. 3. Mapping the active site of papain; specific peptide inhibitors of papain.关于蛋白酶的活性位点。3. 木瓜蛋白酶活性位点的图谱绘制；木瓜蛋白酶的特异性肽抑制剂。
Biochem Biophys Res Commun. 1968 Sep 6;32(5):898-902. doi: 10.1016/0006-291x(68)90326-4.
4
Structure of papain.木瓜蛋白酶的结构。
Nature. 1968 Jun 8;218(5145):929-32. doi: 10.1038/218929a0.
5
The location of the active-site histidine residue in the primary sequence of papain.木瓜蛋白酶一级序列中活性位点组氨酸残基的位置。
Biochem J. 1968 Aug;108(5):861-6. doi: 10.1042/bj1080861.
6
Evidence for histidine in the active site of papain.木瓜蛋白酶活性位点中组氨酸的证据。
Biochem J. 1968 Aug;108(5):855-9. doi: 10.1042/bj1080855.
7
A reinvestigation of residues 64-68 and 175 in papain. Evidence that residues 64 and 175 are asparagine.木瓜蛋白酶中64 - 68位和175位残基的重新研究。证明64位和175位残基为天冬酰胺的证据。
Biochem J. 1970 Feb;116(4):689-92. doi: 10.1042/bj1160689.
8
The amino acid sequence around the active-site cysteine and histidine residues, and the buried cysteine residue in ficin.无花果蛋白酶活性位点半胱氨酸和组氨酸残基周围的氨基酸序列，以及埋藏的半胱氨酸残基。
Biochem J. 1970 Apr;117(2):333-40. doi: 10.1042/bj1170333.
9
Reactivities of the various protonic states in the reactions of papain and of L-cysteine with 2,2'- and with 4,4'- dipyridyl disulphide: evidence for nucleophilic reactivity in the un-ionized thiol group of the cysteine-25 residue of papain occasioned by its interaction with the histidine-159-asparagine-175 hydrogen-bonded system.木瓜蛋白酶和L-半胱氨酸与2,2'-二吡啶二硫化物和4,4'-二吡啶二硫化物反应中各种质子态的反应活性：木瓜蛋白酶中半胱氨酸-25残基的未电离硫醇基团因其与组氨酸-159-天冬酰胺-175氢键系统相互作用而产生亲核反应活性的证据。
Biochem J. 1972 Jun;128(2):471-4. doi: 10.1042/bj1280471.
10
Investigation of the active site of papain with fluorescent probes.用荧光探针研究木瓜蛋白酶的活性位点。
Biochem J. 1973 Aug;133(4):679-86. doi: 10.1042/bj1330679.
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