Regulation of thiocyanate hydrolase activity of sulfur oxidizing bacterium

研究了硫氰酸盐水解酶在硫氰酸硫杆菌细胞中的酶水平变化以及不同化学物质对硫氰酸盐水解酶的抑制反应，以表征该细菌对硫氰酸盐的降解活性。硫代硫酸盐的存在强烈地抑制了该细菌对硫氰酸盐的降解，硫代硫酸盐是该细菌自养能源的良好底物。在含有硫氰酸盐的培养液中加入硫代硫酸盐降低了体内硫氰酸盐水解酶的细胞水平，这是通过抗硫氰酸盐水解酶亚单位蛋白的抗体来估计的。加入硫代硫酸盐抑制了酶蛋白免疫沉淀后[^&lt；14&gt；C]Leu的掺入。这些结果表明，硫代硫酸盐抑制了硫氰酸盐水解酶蛋白的从头合成。进一步的基因克隆和分子分析有助于了解硫代硫酸盐对硫氰酸盐水解酶蛋白合成的抑制作用。氰化物对硫氰酸盐水解酶的活性有强烈的抑制作用，这种抑制作用与硫氰酸盐是非竞争性的，并通过凝胶过滤恢复。羟胺、硼氢化物和结构类似于硫氰酸盐和氰化物的TNBC也抑制了酶的活性。这些化合物似乎与硫氰酸盐水解酶的活性中心不同。此外，硼氢化物与酶的反应部位不同于氰化物和TNBC，氰化物和TNBC在很低的浓度下都对酶有抑制作用，而且它们的结构与酶底物相似，因此这些化合物被认为是与一个对酶活性有重要作用的部位反应，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.