Recombinant expression of rat glycine N-methyltransferase and evidence for contribution of N-terminal acetylation to co-operative binding of S-adenosylmethionine
Recombinant expression of rat glycine N-methyltransferase and evidence for contribution of N-terminal acetylation to co-operative binding of S-adenosylmethionine
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DOI：
10.1042/bj3270407
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发表时间：
1997-10-15
影响因子：
4.1
通讯作者：
Fujioka, M
中科院分区：
文献类型：
--
作者：
Ogawa, H;Gomi, T;Fujioka, M
构建了大鼠甘氨酸N-甲基转移酶在大肠杆菌中的表达载体。重组甘氨酸N-甲基转移酶纯化到同质的DEAE-纤维素和凝胶过滤层析，产量超过80毫克纯酶从1升的培养物。胰蛋白酶肽的HPLC和分离肽的分析表明，重组酶与肝酶的结构相同，除了没有N-末端封闭。大鼠甘氨酸N-甲基转移酶的α-氨基被乙酰化阻断[Ogawa，Konishi，Takata，Nakashima and Fujioka（1987）fur. 168，141-151]。与在所有测试的pH值下显示S-腺苷甲硫氨酸的S形动力学的肝酶[Ogawa和Fujioka（1982）J.Biol.Chem.257，3447-3452]相反，重组酶在低pH下显示双曲线动力学，在高pH下显示S形速率行为。希尔系数随pH增加而增加，在该转变中获得8.11的pK（α）。甘氨酸的Vmax和Km值在两种酶之间没有差异。这些结果表明，消除的正电荷在N-末端的乙酰化或去质子化是需要的合作行为。
An expression vector was constructed that produced rat glycine N-methyltransferase in Escherichia coli. Recombinant glycine N-methyltransferase was purified to homogeneity by DEAE-cellulose and gel-filtration chromatography, with a yield of more than 80 mg of pure enzyme from a 1 litre culture. HPLC of tryptic peptides and analysis of isolated peptides showed that the recombinant enzyme was structurally identical with the liver enzyme except for the absence of N-terminal blocking. The alpha-amino group of rat glycine N-methyltransferase is blocked by acetylation [Ogawa, Konishi, Takata, Nakashima and Fujioka (1987) fur. J. Biochem. 168, 141-151]. In contrast with the liver enzyme, which shows sigmoidal kinetics toward S-adenosylmethionine at all pH values tested [Ogawa and Fujioka (1982) J. Biol. Chem. 257, 3447-3452], the recombinant enzyme exhibited hyperbolic kinetics at low pH and sigmoidal rate behaviour at high pH. The Hill coefficient increased with increasing pH and a pK(a) of 8.11 was obtained in this transition. The values of V-max and K-m for glycine were not different between the two enzymes. These results suggest that elimination of the positive charge at the N-terminal end either by acetylation or deprotonation is required for co-operative behaviour.