Villar E, Schuster B, Peterson D, Schirch V
J Biol Chem. 1985 Feb 25;260(4):2245-52.
C1-Tetrahydrofolate synthase is a multifunctional enzyme which catalyzes three reactions in 1-carbon metabolism: 10-formyltetrahydrofolate synthetase; 5,10-methenyltetrahydrofolate cyclohydrolase; 5,10-methylenetetrahydrofolate dehydrogenase. A rapid 1-day purification procedure has been developed which gives 40 mg of pure enzyme from 10 rabbit livers. The 10-formyltetrahydrofolate synthetase activity of this trifunctional enzyme has a specific activity that is 4-fold higher than the enzyme previously purified from rabbit liver. Conditions have been developed for the rapid isolation of a tryptic fragment of the enzyme which contains the methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities. This fragment is a monomer exhibiting a subunit and native molecular weight of 36,000 in most buffers. However, in phosphate buffers the native molecular weight suggests that the fragment is a dimer. Conditions are also given whereby chymotryptic digestion allows the simultaneous isolation from the native enzyme of a large fragment containing the 10-formyltetrahydrofolate synthetase activity and a smaller fragment containing the dehydrogenase and cyclohydrolase activities. The large fragment is a dimer with a subunit molecular weight of 66,000. The small fragment retains all of the dehydrogenase and cyclohydrolase activities of the native enzyme. The large fragment is unstable but retains most of the 10-formyltetrahydrofolate synthetase activity. Km values of substrates for the two fragments are the same as the values for the native enzyme. The 10-formyltetrahydrofolate synthetase activity of the native enzyme requires ammonium or potassium ions for expression of full catalytic activity. The effect of these two ions on the catalytic activity of the large chymotryptic fragment is the same as with the native enzyme. We have shown by differential scanning calorimetry that the native enzyme contains two protein domains which show thermal transitions at 47 and 60 degrees C. Evidence is presented that the two domains are related to the two protein fragments generated by proteolysis of the native enzyme. The larger of the two domains contains the active site for the 10-formyltetrahydrofolate synthetase activity while the smaller domain contains the active site which catalyzes the dehydrogenase and cyclohydrolase reactions. Replacement of sodium ion buffers with either ammonium or potassium ions results in an increase in stability of the large domain of the native enzyme. This change in stability is not accompanied by a change in the quaternary structure of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)
C1-四氢叶酸合酶是一种多功能酶，它在一碳代谢中催化三个反应：10-甲酰四氢叶酸合成酶；5,10-亚甲基四氢叶酸环水解酶；5,10-亚甲基四氢叶酸脱氢酶。已开发出一种快速的1天纯化程序，可从10个兔肝脏中获得40毫克纯酶。这种三功能酶的10-甲酰四氢叶酸合成酶活性的比活性比先前从兔肝脏中纯化的酶高4倍。已开发出条件用于快速分离该酶的胰蛋白酶片段，该片段含有亚甲基四氢叶酸脱氢酶和亚甲基四氢叶酸环水解酶活性。在大多数缓冲液中，该片段是一种单体，其亚基和天然分子量为36,000。然而，在磷酸盐缓冲液中，天然分子量表明该片段是二聚体。还给出了条件，通过这些条件胰凝乳蛋白酶消化可同时从天然酶中分离出含有10-甲酰四氢叶酸合成酶活性的大片段和含有脱氢酶和环水解酶活性的较小片段。大片段是一种二聚体，亚基分子量为66,000。小片段保留了天然酶的所有脱氢酶和环水解酶活性。大片段不稳定，但保留了大部分10-甲酰四氢叶酸合成酶活性。两个片段底物的Km值与天然酶的值相同。天然酶的10-甲酰四氢叶酸合成酶活性需要铵离子或钾离子来表达完全的催化活性。这两种离子对大的胰凝乳蛋白酶片段催化活性的影响与对天然酶的影响相同。我们通过差示扫描量热法表明，天然酶含有两个蛋白质结构域，它们在47和60摄氏度时显示热转变。有证据表明这两个结构域与天然酶蛋白水解产生的两个蛋白质片段有关。两个结构域中较大的一个含有10-甲酰四氢叶酸合成酶活性的活性位点，而较小的结构域含有催化脱氢酶和环水解酶反应的活性位点。用铵离子或钾离子取代钠离子缓冲液会导致天然酶大结构域稳定性增加。这种稳定性的变化并不伴随着酶四级结构的变化。（摘要截断于400字）
