Regulation of thiocyanate hydrolase activity of sulfur oxidizing bacterium

研究了硫代硫杆菌细胞中硫氰酸酯水解酶的酶水平变化及不同化学物质对硫氰酸酯水解酶的抑制作用，以表征该细菌的硫氰酸酯降解活性。硫代硫酸盐是该细菌自养能量来源的良好底物，它的存在强烈抑制了该细菌对硫氰酸盐的降解。在含硫氰酸盐的培养基中添加硫代硫酸盐可降低体内硫氰酸盐水解酶的细胞水平，这是通过对硫氰酸盐水解酶亚基蛋白的抗体来估计的。添加硫代硫酸盐可抑制酶蛋白免疫沉淀后[^<14>C]亮氨酸的掺入。这些结果表明，硫代硫酸盐抑制硫氰酸酯水解酶蛋白的从头合成。进一步对该基因进行克隆和分子分析，有助于了解硫代硫酸盐对硫氰酸酯水解酶蛋白合成的抑制作用。氰化物强烈抑制硫氰酸酯水解酶的活性，这种抑制作用对硫氰酸酯没有竞争性，并通过凝胶过滤恢复。羰基试剂，如羟胺、硼氢化物和硫氰酸盐和氰化物的结构类似物TNBC，也会抑制酶的活性。这些化合物似乎与硫氰酸酯水解酶活性中心不同的位点发生反应。此外，硼氢化物与该酶的反应位点不同于氰化物和三溴化bc。氰化物和TNBC都在很低的浓度下抑制了酶的活性，并且它们与酶底物具有类似的结构，因此这些化合物被认为与一个特定的位点发生反应，该位点对酶的活性具有重要的作用，位于酶蛋白的活性中心附近。

Changes of the enzyme level of thiocyanate hydrolase in Thiobacillus thioparus cells and inhibitory reaction of various chemicals to thiocyanate hydrolase were investigated to characterize thiocyanate degrading activity of the bacteria. Thiocyanate degradation by this bacterium was strongly inhibited by the presence of thiosulfate that is a good substrate for autotrophic energy source for this bacterium. Addition of thiosulfate in culture medium containing thiocyanate decreased cellular level of thiocyanate hydrolase in vivo, which were estimated by using antibodies against subunit proteins of thiocyanate hydrolase. Incorporation of [^<14>C] Leu following immunoprecipitation of the enzyme protein was suppressed by the addition of thiosulfate. These results suggested that thiosulfate inhibited de novo synthesis of thiocyanate hydrolase proteins. Further experiment on gene cloning and molecular analysis of the gene is useful to understand inhibitory effect of thiosulfate on the protein systhesis of thiocyanate hydrolase.Cyanide strongly inhibited the activity of thiocyanate bydrolase, Which inhibition was noncompetitive to thiocyanate and was restored by a gel filtration. Carbonyl reagents, such as hydroxylamine, borohydride, and TNBC that is a structural analog for thiocyanate and cyanide, also inhibited the enzyme activity. These compounds seemed to react with a site different from the active center of the thiocyanate hydrolase. Furthermore reaction site of borohydride with the enzyme was different from those of cyanide and TNBC.Both cyanide and TNBC inhibited the enzyme activity at quite low concentration, and they have analogous structure to the enzyme substrate, therefore these compounds are thouht to react with a specific site that has an important function to manifest the enzyme activity, locating at close to the active center of the enzyme protein.