苯并呋咱作为一种硫醇特异性反应探针。其与木瓜蛋白酶、无花果蛋白酶、菠萝蛋白酶及低分子量硫醇反应的动力学。
Benzofuroxan as a thiol-specific reactivity probe. Kinetics of its reactions with papain, ficin, bromelain and low-molecular-weight thiols.
作者信息
Shipton M, Brocklehurst K
出版信息
Biochem J. 1977 Dec 1;167(3):799-810. doi: 10.1042/bj1670799.
Abstract
- The characteristics of benzofuroxan (benzofurazan 1-oxide, benzo-2-oxa-1,3-diazole N-oxide) that relate to its application as a reactivity probe for the study of environments of thiol groups are discussed. 2. To establish a kinetic and mechanistic basis for its use as a probe, a kinetic study of its reaction with 2-mercaptoethanol was carried out. 3. This reaction appears to proceed by a rate-determining attack of the thiolate ion on one of the electrophilic centres of benzofuroxan (possibly C-6) to provide a low steady-state concentration of an intermediate adduct; rapid reaction of this adduct with a second molecule of thiol gives the disulphide and o-benzoquinone dioxime. 4. The effects of the different types of environment that proteins can provide on the kinetic characteristics of reactions of thiol groups with benzofuroxan are delineated. 5. Benzofuroxan was used as a thiolspecific reactivity probe to investigate the active centres of papain (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). The results support the concept that the active centres of all three enzymes either contain a nucleophilic thiolate ion whose formation is characterized by a pKa of 3-4 and whose reaction with an electrophile can be assisted by interaction of a site of high electron density in the electrophile with active-centre imidazolium ion of pKa 8-9, or can provide such ions by protonic redistribution in enzyme-reagent or enzyme-substrate complexes.
摘要
- 讨论了苯并呋咱（苯并呋喃 -1- 氧化物，苯并 -2- 恶唑 -1,3- 二氮杂环氧化物）作为研究巯基环境的反应性探针的相关特性。2. 为了建立其作为探针使用的动力学和机理基础，对其与 2- 巯基乙醇的反应进行了动力学研究。3. 该反应似乎是通过硫醇盐离子对苯并呋咱的一个亲电中心（可能是 C-6）的速率决定攻击来进行的，以提供低稳态浓度的中间加合物；该加合物与第二个硫醇分子的快速反应生成二硫化物和邻苯醌二肟。4. 阐述了蛋白质所能提供的不同类型环境对巯基与苯并呋咱反应动力学特性的影响。5. 苯并呋咱用作巯基特异性反应性探针，研究了木瓜蛋白酶（EC 3.4.22.2）、无花果蛋白酶（EC 3.4.22.3）和菠萝蛋白酶（EC 3.4.22.4）的活性中心。结果支持了这样的概念，即所有这三种酶的活性中心要么含有一个亲核硫醇盐离子，其形成的特征 pKa 为 3 - 4，其与亲电试剂的反应可通过亲电试剂中高电子密度位点与 pKa 为 8 - 9 的活性中心咪唑鎓离子的相互作用来促进，要么可以通过酶 - 试剂或酶 - 底物复合物中的质子重新分布来提供这样的离子。
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1
Benzofuroxan as a thiol-specific reactivity probe. Kinetics of its reactions with papain, ficin, bromelain and low-molecular-weight thiols.苯并呋咱作为一种硫醇特异性反应探针。其与木瓜蛋白酶、无花果蛋白酶、菠萝蛋白酶及低分子量硫醇反应的动力学。
Biochem J. 1977 Dec 1;167(3):799-810. doi: 10.1042/bj1670799.
2
Evaluation of benzofuroxan as a chromophoric oxidizing agent for thiol groups by using its reactions with papain, ficin, bromelain and low-molecular-weight thiols.通过苯并呋咱与木瓜蛋白酶、无花果蛋白酶、菠萝蛋白酶及低分子量硫醇的反应来评估其作为硫醇基团发色氧化剂的性能。
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8
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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本文引用的文献
1
Gamma radiolysis of cystine in aqueous solution. Dose-rate effects and a proposed mechanism.水溶液中胱氨酸的γ辐解。剂量率效应及一种提出的机制。
J Am Chem Soc. 1967 Jan 18;89(2):226-8. doi: 10.1021/ja00978a007.
2
The reactivity of the sulfhydryl groups of lobster muscle glyceraldehyde 3-phosphate dehydrogenase.龙虾肌肉磷酸甘油醛脱氢酶巯基的反应活性
Biochemistry. 1969 Mar;8(3):1076-82. doi: 10.1021/bi00831a039.
3
Stereospecific alkylation with asymmetric reagents.使用不对称试剂进行立体专一性烷基化反应。
Eur J Biochem. 1968 Jan;3(3):267-75. doi: 10.1111/j.1432-1033.1968.tb19526.x.
4
Glutathione. II. Chemical aspects of azoester procedure for oxidation to disulfide.谷胱甘肽。二。偶氮酯氧化成二硫化物过程的化学方面。
Biochim Biophys Acta. 1969 Oct 7;192(1):8-14. doi: 10.1016/0304-4165(69)90003-8.
5
Reaction of the sulfhydryl group of papain with chloroacetic acid.木瓜蛋白酶的巯基与氯乙酸的反应。
J Biol Chem. 1969 Oct 10;244(19):5095-9.
6
Reaction of papain with -bromo- -(5-imidazolyl)propionic acid.木瓜蛋白酶与β-溴-β-(5-咪唑基)丙酸的反应。
Biochemistry. 1972 Jan 18;11(2):164-9. doi: 10.1021/bi00752a005.
7
On the mode of activation of the catalytically essential sulfhydryl group of papain.关于木瓜蛋白酶催化必需巯基的激活模式。
Eur J Biochem. 1973 Feb 15;33(1):104-9. doi: 10.1111/j.1432-1033.1973.tb02660.x.
8
Mercaptide-imidazolium ion-pair: the reactive nucleophile in papain catalysis.硫醇盐 - 咪唑鎓离子对：木瓜蛋白酶催化中的活性亲核试剂。
FEBS Lett. 1974 Oct 1;47(1):15-8. doi: 10.1016/0014-5793(74)80415-1.
9
The unreliability of estimates of group dissociation constants.基团解离常数估计值的不可靠性。
Biochem J. 1976 Mar 1;153(3):627-9. doi: 10.1042/bj1530627.
10
Evaluation of benzofuroxan as a chromophoric oxidizing agent for thiol groups by using its reactions with papain, ficin, bromelain and low-molecular-weight thiols.通过苯并呋咱与木瓜蛋白酶、无花果蛋白酶、菠萝蛋白酶及低分子量硫醇的反应来评估其作为硫醇基团发色氧化剂的性能。
Biochem J. 1977 Mar 1;161(3):627-37. doi: 10.1042/bj1610627.
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