Cacciapuoti G, Porcelli M, Cartenì-Farina M, Gambacorta A, Zappia V
Eur J Biochem. 1986 Dec 1;161(2):263-71. doi: 10.1111/j.1432-1033.1986.tb10442.x.
The enzyme propylamine transferase, catalyzing the transfer of the propylamine moiety from S-adenosyl(5')-3-methylthiopropylamine to several amine acceptors, has been purified 643-fold in 20% yield from Sulfolobus solfataricus, an extreme thermophilic archaebacterium optimally growing at 87 degrees C. The purified enzyme (specific activity 2.05 units/mg protein), is homogeneous by criteria of gel electrophoresis, gel filtration, isoelectric focusing and ultracentrifugation analysis. The molecular mass of the native enzyme was estimated to be about 110 kDa by gel permeation and ultracentrifugation analysis. The protein migrates on SDS/polyacrylamide gel electrophoresis as a single band of 35 kDa, suggesting that the enzyme is a trimer composed by identical subunits. An optimum pH of 7.5 and an acidic isoelectric point of 5.3 have been calculated. The optimum temperature was 90 degrees C and no loss of activity is observable even after exposure of the purified enzyme to 100 degrees C for 1 h. No reducing agents are required for enzymatic activity. Substrate specificity towards the amine acceptors is rather broad in that 1,3-diaminopropane (Km = 1675 microM), putrescine (Km = 3850 microM), sym-norspermidine (Km = 954 microM) and spermidine (Km = 1539 microM) are recognized as substrates. Conversely S-adenosyl(5')-3-methylthiopropylamine is the only propylamine donor (Km = 7.9 microM) and the deamination of the sulfonium compound prevents the recognition by the enzyme. The reaction is irreversible and initial-rate kinetic studies indicate that the propylamine transfer is operated through a sequential mechanism. 5'-Methylthioadenosine, a product of the reaction, acts as a powerful competitive inhibitor with a Ki of 3.7 microM. Enzyme-substrate binding sites have been investigated with the aid of several substrate analogs and products. Among the compounds assayed, 5'-methylthiotubercidin, S-adenosyl(5')-3-thiopropylamine and S-adenosyl-3-thio-1,8-diaminooctane are the most active inhibitors.
丙胺转移酶可催化丙胺部分从S-腺苷基（5'）-3-甲基硫丙胺转移至多种胺受体。该酶已从嗜热栖热菌（一种在87摄氏度下最佳生长的极端嗜热古细菌）中以20%的产率纯化了643倍。通过凝胶电泳、凝胶过滤、等电聚焦和超速离心分析等标准，纯化后的酶（比活性为2.05单位/毫克蛋白质）是均一的。通过凝胶渗透和超速离心分析，天然酶的分子量估计约为110 kDa。该蛋白在SDS/聚丙烯酰胺凝胶电泳上迁移为一条35 kDa的单带，表明该酶是由相同亚基组成的三聚体。已计算出最佳pH值为7.5，酸性等电点为5.3。最佳温度为90摄氏度，即使将纯化后的酶在100摄氏度下暴露1小时，也未观察到活性损失。酶活性不需要还原剂。对胺受体的底物特异性相当广泛，因为1,3-二氨基丙烷（Km = 1675 microM）、腐胺（Km = 3850 microM）、对称-去甲亚精胺（Km = 954 microM）和亚精胺（Km = 1539 microM）都被识别为底物。相反，S-腺苷基（5'）-3-甲基硫丙胺是唯一的丙胺供体（Km = 7.9 microM），并且该锍化合物的脱氨作用会阻止酶对其的识别。该反应是不可逆的，初始速率动力学研究表明丙胺转移是通过顺序机制进行的。反应产物5'-甲硫基腺苷作为一种强大的竞争性抑制剂，Ki为3.7 microM。借助几种底物类似物和产物对酶-底物结合位点进行了研究。在所检测的化合物中，5'-甲硫基结核菌素、S-腺苷基（5'）-3-硫丙胺和S-腺苷基-3-硫-1,8-二氨基辛烷是最有效的抑制剂。
