Santha R, Savithri H S, Rao N A, Vaidyanathan C S
Department of Biochemistry, Indian Institute of Science, Bangalore.
Eur J Biochem. 1995 May 15;230(1):104-10.
2,3-Dihydroxybenzoic acid decarboxylase, the last enzyme in the fungal metabolism of indole to catechol, catalyzes the non-oxidative decarboxylation of 2,3-dihydroxybenzoic acid to catechol. Unlike most other decarboxylases, this enzyme does not require a cofactor, underlining the importance of active-site residues in the reaction mechanism. Earlier studies from this laboratory [Kamath, A. V., Appaji Rao, N. & Vaidyanathan, C. S. (1989) Biochem. Biophys. Res. Commun. 165, 20-26], have shown that the sulfhydryl agent N-ethylmaleimide (MalNEt) inactivated the enzyme by modifying a single class of cysteine residues and that this inactivation was prevented in the presence of salicylate, a substrate analogue. In the present study, this essential cysteine residue has been identified by specific labelling with [14C]-MalNEt using the differential labelling technique. The stoichiometry of incorporation of [14C]MalNEt was approximately one/subunit of the homotetrameric protein. The peptide bearing this reactive cysteine residue was isolated by tryptic digestion of the differentially labelled enzyme and subsequent reverse-phase chromatography of the peptide mixture. The sequence of the major radioactive peptide that was identified to be the active-site peptide, was LLGLAETCK. A search for sequences similar to this active-site peptide indicated that this sequence was probably unique to the decarboxylase under study. A partial primary structure map constructed from the sequences of peptides derived from enzymic cleavage of the protein using endoproteinase Glu-C and trypsin did not share any significant sequence similarity with sequences reported in the database, again suggesting the uniqueness of the enzyme. This is the first report on the active-site peptide and the partial primary structure of a non-oxidative decarboxylase catalyzing the removal of a carboxyl group from an aromatic nucleus.
2,3 -二羟基苯甲酸脱羧酶是真菌将吲哚代谢为儿茶酚过程中的最后一种酶，它催化2,3 -二羟基苯甲酸非氧化脱羧生成儿茶酚。与大多数其他脱羧酶不同，这种酶不需要辅因子，这突出了活性位点残基在反应机制中的重要性。本实验室早期的研究[卡马特，A. V.，阿帕吉·拉奥，N.和瓦伊迪亚纳坦，C. S.（1989年）《生物化学与生物物理研究通讯》165，20 - 26]表明，巯基试剂N -乙基马来酰亚胺（MalNEt）通过修饰一类单一的半胱氨酸残基使该酶失活，并且在底物类似物水杨酸盐存在的情况下这种失活被阻止。在本研究中，使用差异标记技术通过用[14C] - MalNEt进行特异性标记鉴定出了这个必需的半胱氨酸残基。[14C]MalNEt的掺入化学计量约为同四聚体蛋白的一个/亚基。通过对差异标记的酶进行胰蛋白酶消化以及随后对肽混合物进行反相色谱法，分离出了带有这个反应性半胱氨酸残基的肽。被鉴定为活性位点肽的主要放射性肽的序列是LLGLAETCK。对与该活性位点肽相似序列的搜索表明，这个序列可能是所研究的脱羧酶特有的。使用内肽酶Glu - C和胰蛋白酶对该蛋白质进行酶解得到的肽序列构建的部分一级结构图谱与数据库中报道的序列没有任何显著的序列相似性，这再次表明了该酶的独特性。这是关于催化从芳环上去除羧基的非氧化脱羧酶的活性位点肽和部分一级结构的首次报道。
