Cysteine conjugate β-lyase activity of amino acid decarboxylases.
Cysteine conjugate β-lyase activity of amino acid decarboxylases.
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DOI：
10.1042/bst026s269
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发表时间：
1998-08-01
影响因子：
3.9
通讯作者：
Teesdale-Spittle, PH
中科院分区：
文献类型：
--
作者：
Buckberry, LD;Patel, R;Teesdale-Spittle, PH
酶介导的L-半胱氨酸缀合物的裂解导致化学计量量的丙酮酸盐、氨和反应性硫醇物质的产生。已经确定，这种硫醇物质是暴露于某些外源性物质后发生的致突变和细胞毒性后果的原因[1]。已证明CCBL的生理作用是氨基酸的转氨作用或CC裂解作用[2，3，4]。本文报道L-谷氨酸脱羧酶（C。perfingem）、L-精氨酸脱羧酶（E. coli）、L-苯丙氨酸脱羧酶（S. fueculis）、L-酪氨酸脱羧酶（S. fueculis）和L-鸟氨酸脱羧酶（E. coli）对CCBL最适底物DCVC具有CCBL活性。还在辅因子PLP存在下测定CCBL活性，并根据McKinney等人的方法合成PLP依赖性酶抑制剂AOAA DCVC。（1959）[5]。脱羧酶作为冻干制剂从Sigma Chemical Co. Ltd.（Poole，Dorset，UK）和Fluka Chemicals（吉灵厄姆，Dorset，UK）。所用的所有其他试剂均为分析级，购自Sigma Chemical Co. Ltd.。（Poole，Dorset，UK）。根据Laemmli的方法[6]，通过考马斯亮蓝染色的SDSPAGE显示脱羧酶制剂是纯的。使用Gutman和Wahlefeld（1974）的方法测定CCBL活性（产生的nmol丙酮酸盐/min”mg-1蛋白）[7]。使用的DCVC浓度为7.5 mM终浓度。测定天冬氨酸氨基转移酶对DCVC的CCBL活性作为对照值。所有测试的脱羧酶都显示对DCVC具有CCBL活性（表1）。结果表明，最适酶为L-谷氨酸脱羧酶（ASAT的30%），其次为鸟氨酸脱羧酶（20%）、L-精氨酸脱羧酶（14%）、L-苯丙氨酸脱羧酶（1.3%）、L-酪氨酸脱羧酶（10%）。在PLP（2 mM）存在下，L-谷氨酸脱羧酶、L-精氨酸脱羧酶和L-苯丙氨酸脱羧酶的活性分别增加2、1.6和1.8倍（表2）。ASAT和酪氨酸脱羧酶的活性没有显著增加。在KMB（10 mM）的存在下，L-谷氨酸脱羧酶的活性没有显着改变。ASAT和L-精氨酸脱羧酶的活性分别提高了1.7和2.2倍，而L-苯丙氨酸脱羧酶和L-酪氨酸脱羧酶的活性降低。
Enzyme mediated p-lysis of L-cysteine conjugates results in the production of stoichiometric amounts of pyruvate, ammonia and a reactive thiolic species. It has been well established that this thiolic species is responsible for the mutagenic and cytotoxic consequences which occur following exposure to certain xenobiotics [l]. The physiological role of CCBL's has been demonstrated to be that of transamination or CC lysis of amino acids [2, 3, 4]. Here we report that L-glutamic acid decarboxylase (C. perfingem), L-arginine decarboxylase (E. coli), L-phenylalanine decarboxylase (S. fueculis), L-tyrosine decarboxylase (S. fueculis) and L-omithine decarboxylase (E. coli) exhibit CCBL activity towards DCVC, the optimum CCBL substrate. CCBL activity was also determined in the presence of the cofactor PLP and the PLP dependent enzyme inhibitor AOAA DCVC was synthesised according to the method of McKinney et al.(1959)[5]. Decarboxylase enzymes were obtained as lyophilised preparations from Sigma Chemical Co. Ltd.(Poole, Dorset, UK) and Fluka Chemicals (Gillingham, Dorset, UK). All other reagents used were of analytical grade and obtained from Sigma Chemical Co. Ltd.(Poole, Dorset, UK). The decarboxylase preparations were shown to be pure by SDSPAGE with Coomassie Brilliant Blue staining according to the method of Laemmli [6]. CCBL activity (nmol pyruvate produced min" mg-'protein) was determined using the method of Gutman and Wahlefeld (1974)[7]. The DCVC concentration used was 7.5 mM final concentration. The CCBL activity of aspartate amino transferase towards DCVC was determined as a control value.All the decarboxylase enzymes tested were shown to have CCBL activity towards DCVC (Table 1). L-glutamic acid decarboxylase was the optimum CCBL (30% of ASAT), followed in descending order by omithine decarboxylase (20%), L-arginine decarboxylase (14%), L-phenylalanine decarboxylase (1 3%), L-tyrosine decarboxylase (10%). In the presence of PLP (2mM) the activity of L-glutamic acid decarboxylase, L-arginine decarboxylase and L-phenylalanine decarboxylase increased 2, 1.6 and 1.8 fold, respectively (Table 2). The activity of ASAT and tyrosine decarboxylase was not significantly increased. In the presence of KMB (1OmM) the activity of L-glutamic acid decarboxylase was not significantly altered. The activity of ASAT and L-arginine decarboxylase was increased 1.7 and 2.2 fold, respectively, however, the activity of L-phenylalanine decarboxylase and L-tyrosine decarboxylase decreased.