Krone U E, McFarlan S C, Hogenkamp H P
Department of Biochemistry, University of Minnesota, Minneapolis 55455.
Eur J Biochem. 1994 Mar 15;220(3):789-94. doi: 10.1111/j.1432-1033.1994.tb18680.x.
A protein catalyzing the tritium exchange of [5-3H]deoxyuridine monophosphate ([5-3H]dUMP) for solvent protons and the dehalogenation of 5-bromo-deoxyuridine monophosphate (Br-dUMP) has been isolated from the methanogenic archaea Methanobacterium thermoautotrophicum. These two activities are well-established side reactions of thymidylate synthase and do not require cofactors. Sodium dodecylsulfate/polyacrylamide gel electrophoresis of the purified enzyme showed a single band with a molecular mass of 27 kDa. The suggested molecular mass of the native protein calculated from sedimentation equilibrium experiments was 33.5 kDa, indicating that the enzyme is a monomer. The pH optima were 9.0 and 7.0 for the exchange reaction and the dehalogenation, respectively. The effects of temperature, salt, reducing agent and inhibitors were determined. The apparent Km for the tritium exchange from [5-3H]dUMP was 7 microM and for the dehalogenation of Br-dUMP was 14 microM. However, thus far, the conditions for dTMP synthesis from dUMP have not yet been established. Incubation of the enzyme with dUMP, tetrahydromethanopterin, a folate analog present in methanogens, and formaldehyde did not yield dTMP. The first 30 amino acids of the amino terminus have been sequenced. However, there is no similarity with any of the thymidylate synthases. Surprisingly, the protein from M. thermoautotrophicum appears to be related to chitin synthases from several organisms.
一种能催化[5-³H]脱氧尿苷单磷酸（[5-³H]dUMP）与溶剂质子进行氚交换以及5-溴脱氧尿苷单磷酸（Br-dUMP）脱卤反应的蛋白质已从产甲烷古菌嗜热自养甲烷杆菌中分离出来。这两种活性是胸苷酸合成酶公认的副反应，且不需要辅因子。纯化酶的十二烷基硫酸钠/聚丙烯酰胺凝胶电泳显示有一条分子量为27 kDa的单一蛋白条带。根据沉降平衡实验计算出的天然蛋白质推测分子量为33.5 kDa，表明该酶是单体。交换反应和脱卤反应的最适pH分别为9.0和7.0。测定了温度、盐、还原剂和抑制剂的影响。[5-³H]dUMP氚交换的表观Km为7 μM，Br-dUMP脱卤的表观Km为14 μM。然而，到目前为止，由dUMP合成dTMP的条件尚未确定。将该酶与dUMP、四氢甲蝶呤（产甲烷菌中存在的一种叶酸类似物）和甲醛一起孵育未产生dTMP。已对氨基末端的前30个氨基酸进行了测序。然而，与任何胸苷酸合成酶均无相似性。令人惊讶的是，嗜热自养甲烷杆菌的这种蛋白质似乎与几种生物的几丁质合成酶有关。
