Inhibition of thiaminase I from Bacillus thiaminolyticus. Evidence supporting a covalent 1,6-dihydropyrimidinyl-enzyme intermediate.
Inhibition of thiaminase I from Bacillus thiaminolyticus. Evidence supporting a covalent 1,6-dihydropyrimidinyl-enzyme intermediate.
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抑制硫胺素解芽孢杆菌的硫胺素酶 I。
DOI：
10.1021/bi00381a028
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Slama,JT
中科院分区：
文献类型：
--
作者：
Hutter,JA;Slama,JT
美国德克萨斯大学健康科学中心生物化学系，圣安东尼奥，德克萨斯州78284-7760摘要：来自Bacillus thiaminolyticus菌株Matsukawa et Misawa的Thiaminase I被4-氨基-6-氯-2-甲基嘧啶完全不可逆地抑制。抑制是一个依赖于时间的一级过程，抑制剂浓度为5mm时的半衰期为4小时。在底物硫胺素和喹啉的存在下保护酶活性，以及观察到在失活过程中释放出化学量的无机氯化物，支持了特定的活性位点定向失活。4-氨基-5-（苯胺甲基）-6-氯-2-甲基嘧啶类似于硫胺素与苯胺碱交换产物的结构，使硫胺素酶的失活速度快约2个数量级。失活也是完全不可逆的，并且是一个与时间相关的一阶过程，在低抑制剂浓度下表现出饱和（a,= 96µ）。酶失活可以解释为在酶活性位点的亲核试剂取代氯-氯嘧啶的结果。这种失活表明亚硫酸氢盐催化硫胺素裂解的Zoltewicz-Kauffman模型[Zoltewicz， J. A., & Kauffman， G. M.(1977)]。化学。[Soc. 99, 3134-3142]，需要在硫胺素嘧啶环的1,6双键上可逆的亲核加成催化剂，这也可能适用于硫胺素酶。层合酶是通过在嘧啶甲基和噻唑之间切割维生素来破坏维生素的酶（Fujita, 1954; Murata, 1965, 1982）。硫胺酶I （EC 2.5）1.2)催化碱交换反应，在该反应中，硫胺素的（4-氨基-2-甲基-5-嘧啶基）甲基被转移到多种有机亲核试剂中的任何一种，如苯胺、喹啉、吡啶和半胱氨酸。一个相关的活性，硫胺酶II，催化嘧啶基甲基的转移。由美国国立卫生研究院（GM 32821）和Robert A. Welch基金会（AQ-979）的研究经费支持。摘自JAH的博士论文，专门针对水。图1为硫胺素酶I催化的硫胺素与苯胺的反应。硫胺酶I存在于蕨类、鱼类、软体动物、甲壳类和微生物等多种生物中（Fujita, 1954; Murata, 1965, 1982）。虽然该酶的生物学功能尚不清楚，但它具有一定的兽医重要性，因为已经证明，喂食含有过量该酶的饲料的动物会出现硫胺素缺乏症的神经症状（Green et al., 1941; Woolley, 1941）。瘤胃微生物产生硫胺酶被认为是家畜某些硫胺素缺乏的原因(Edwin & Jackson, 1970; Edwin et al.；
Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78284-7760 Received August 19, 1986; Revised Manuscript Received December 4, 1986 abstract: Thiaminase I from Bacillus thiaminolyticus strain Matsukawa et Misawa is completely and irreversibly inhibited by treatment with 4-amino-6-chloro-2-methylpyrimidine. Inhibition is a time-dependent first-order process, exhibiting a half-time of 4 h at an inhibitor concentration of 5 mM. A specific ac-tive-site-directed inactivation is supported by protection of the enzymatic activity in the presence of the substrates thiamin and quinoline as well as by the observation that a stoichiometric amount of inorganic chloride is released during inactivation. 4-Amino-5-(anilinomethyl)-6-chloro-2-methylpyrimidine, which resembles the structure of the product of base exchange of thiamin with aniline, inactivates thiaminase approximately 2 orders of magnitude faster. Inactivation is again complete and irreversible and is a time-dependent first-order process, in this case exhibiting saturation at low inhibitor concentrations (A,= 96 µ). Enzyme inactivation can be explained as the result of displacement of chloride from the chlo-ropyrimidine by a nucleophile at the enzyme active site. The inactivation suggests that the Zoltewicz-Kauffman model of bisulfite-catalyzed thiamin cleavage [Zoltewicz, J. A., & Kauffman, G. M.(1977) J. Am. Chem. Soc. 99, 3134-3142], which calls for the reversible nucleophilic addition of catalyst across the 1, 6 double bond of thiamin’s pyrimidine ring, may be applicable to thiaminase as well. laminases are enzymes that destroy thiamin bycleaving the vitamin between the pyrimidinylmethyl group and the thiazole (Fujita, 1954; Murata, 1965, 1982). Thiaminase I (EC 2.5. 1.2) catalyzes a base exchange reaction in which the (4-amino-2-methyl-5-pyrimidinyl) methyl group of thiamin is transferred to any one of a variety of organic nucleophiles, eg, aniline, quinoline, pyridine, and cysteine. A related activity, thiaminase II, catalyzes the transfer of the pyrimidinylmethyl f Supported by research grants to JTS from the National Institutes of Health (GM 32821) and from the Robert A. Welch Foundation (AQ-979). Taken from the Ph. D. dissertation of JAH specifically to water. Scheme I shows the reaction catalyzed by thiaminase I between thiamin and aniline. Thiaminase I occurs in such diverse organisms as ferns, fish, mollusks, Crustacea, and microorganisms (Fujita, 1954; Murata, 1965, 1982). Although the biological function of the enzyme is unknown, it is of some veterinary importance, as it has been demonstrated that animals fed a diet containing excessive quantities of the enzyme develop the neurological symptoms of thiamindeficiency (Green et al., 1941; Woolley, 1941). Production of thiaminase by ruminal microorganisms has been suggested to be the cause of certain thiamin defi-ciencies in livestock (Edwin & Jackson, 1970; Edwin et al.,