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。
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.
作者信息
Malthouse J P, Brocklehurst K
出版信息
Biochem J. 1980 Jan 1;185(1):217-22. doi: 10.1042/bj1850217.
Abstract
- Whereas the second-order rate constants for the reaction of the thiolate ion of 2-mercaptoethanol with 4,4'-dipyridyl disulphide (k4PDS) and with 5,5'-dithiobis-2-nitrobenzoate dianion increase with decreasing dielectric constant of the solvent, or remain unchanged, the rate constant for the analogous reaction with 2,2'-dipyridyl disulphide (k2PDS) decreases. This anomalous solvent effect and other unusual physicochemical properties of 2,2'-dipyridyl disulphide are discussed. 2. The differential effect of solvent on the reactions of thiolate ion with the 2,2'- and 4,4'-dipyridyl disulphides is shown to provide a method of characterizing solvent environments of thiol groups in proteins by a reactivity-probe method that should not suffer from the usual drawback associated with the existence of steric or binding effects of unknown magnitude. Application of the method to ficin (EC 3.4.22.3) suggests that its active-centre thiol group resides in a relatively hydrophobic environment. 3. The pH-k profile for the reaction of ficin with 4,4'-dipyridyl disulphide is reported.
摘要
- 2-巯基乙醇硫醇负离子与4,4'-二吡啶二硫化物（k4PDS）以及与5,5'-二硫代双-2-硝基苯甲酸二价阴离子反应的二级速率常数会随着溶剂介电常数的降低而增大，或者保持不变，而与2,2'-二吡啶二硫化物（k2PDS）发生类似反应的速率常数则会减小。本文讨论了这种反常的溶剂效应以及2,2'-二吡啶二硫化物的其他异常物理化学性质。2. 硫醇负离子与2,2'-和4,4'-二吡啶二硫化物反应时溶剂的差异效应表明，通过一种反应性探针方法可以表征蛋白质中硫醇基团的溶剂环境，该方法不应受到与未知大小的空间或结合效应相关的常见缺点的影响。将该方法应用于无花果蛋白酶（EC 3.4.22.3）表明其活性中心硫醇基团位于相对疏水的环境中。3. 报道了无花果蛋白酶与4,4'-二吡啶二硫化物反应的pH-k曲线。
相似文献
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.
2
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.
3
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.
4
5
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.
6
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.
7
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.
8
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.
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
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.
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Partitioning of a polymer into a nanoscopic protein pore obeys a simple scaling law.聚合物在纳米级蛋白质孔中的分配遵循一个简单的标度律。
Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10137-41. doi: 10.1073/pnas.181089798. Epub 2001 Aug 14.
2
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.
3
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.
本文引用的文献
1
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.
2
Coenzyme B 12 model studies. Equilibria and kinetics of axial ligation of methylaquocobaloxime by thiols.
J Am Chem Soc. 1972 Mar 22;94(6):1894-901. doi: 10.1021/ja00761a018.
3
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.
4
Specific covalent modification of thiols: applications in the study of enzymes and other biomolecules.硫醇的特异性共价修饰：在酶和其他生物分子研究中的应用
Int J Biochem. 1979;10(4):259-74. doi: 10.1016/0020-711x(79)90088-0.
5
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.
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
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