Santha R, Rao N A, Vaidyanathan C S
Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Biochim Biophys Acta. 1996 Apr 16;1293(2):191-200. doi: 10.1016/0167-4838(95)00242-1.
The non-oxidative decarboxylation of aromatic acids is a poorly understood reaction. The transformation of 2,3-dihydroxybenzoic acid to catechol in the fungal metabolism of indole is a prototype of such a reaction. 2,3-Dihydroxybenzoic acid decarboxylase (EC 4.1.1.46) which catalyzes this reaction was purified to homogeneity from anthranilate induced cultures of Aspergillus oryzae using affinity chromatography. The enzyme did not require cofactors like NAD+, PLP, TPP or metal ions for its activity. There was no spectral evidence for the presence of enzyme bound cofactors. The preparation, which was adjudged homogeneous by the criteria of SDS-PAGE, sedimentation analysis and N-terminal analysis, was characterized for its physicochemical and kinetic parameters. The enzyme was inactivated by group-specific modifiers like diethyl pyrocarbonate (DEPC) and N-ethylmaleimide (NEM). The kinetics of inactivation by DEPC suggested the presence of a single class of essential histidine residues, the second order rate constant of inactivation for which was 12.5 M-1 min-1. A single class of cysteine residues was modified by NEM with a second order rate constant of 33 M-1 min-1. Substrate analogues protected the enzyme against inactivation by both DEPC and NEM, suggesting the location of the essential histidine and cysteine to be at the active site of the enzyme. The incorporation of radiolabelled NEM in a differential labelling experiment was 0.73 mol per mol subunit confirming the presence of a single essential cysteine per active-site. Differentially labelled enzyme was enzymatically cleaved and the peptide bearing the label was purified and sequenced. The active-site peptide LLGLAETCK and the N-terminal sequence MLGKIALEEAFALPRFEEKT did not bear any similarity to sequences reported in the Swiss-Prot Protein Sequence Databank, a reflection probably of the unique primary structure of this novel enzyme. The sequences reported in this study will appear in the Swiss-Prot Protein Sequence Databank under the accession number P80402.
芳香酸的非氧化脱羧反应是一个了解甚少的反应。在吲哚的真菌代谢过程中，2,3 - 二羟基苯甲酸转化为儿茶酚就是这类反应的一个实例。利用亲和层析法从米曲霉邻氨基苯甲酸诱导培养物中纯化得到了催化该反应的2,3 - 二羟基苯甲酸脱羧酶（EC 4.1.1.46），使其达到了均一性。该酶的活性不需要诸如NAD⁺、磷酸吡哆醛、焦磷酸硫胺素或金属离子等辅因子。没有光谱证据表明存在与酶结合的辅因子。根据SDS - PAGE、沉降分析和N - 端分析标准判定为均一的该制剂，对其理化参数和动力学参数进行了表征。该酶会被焦碳酸二乙酯（DEPC）和N - 乙基马来酰亚胺（NEM）等基团特异性修饰剂灭活。DEPC灭活动力学表明存在一类单一的必需组氨酸残基，其二级灭活速率常数为12.5 M⁻¹ min⁻¹。NEM修饰了一类单一的半胱氨酸残基，二级速率常数为33 M⁻¹ min⁻¹。底物类似物可保护该酶免受DEPC和NEM的灭活，这表明必需组氨酸和半胱氨酸位于酶的活性位点。在差异标记实验中，每摩尔亚基放射性标记NEM的掺入量为0.73摩尔，证实每个活性位点存在一个单一的必需半胱氨酸。对差异标记的酶进行酶切，纯化并测序带有标记的肽段。活性位点肽段LLGLAETCK和N - 端序列MLGKIALEEAFALPRFEEKT与瑞士蛋白质序列数据库中报道的序列没有任何相似性，这可能反映了这种新型酶独特的一级结构。本研究报道的序列将以登录号P80402出现在瑞士蛋白质序列数据库中。
