N-5-METHYLTETRAHYDROMETHANOPTERIN - COENZYME-M METHYLTRANSFERASE IN METHANOGENIC ARCHAEBACTERIA IS A MEMBRANE-PROTEIN
N-5-METHYLTETRAHYDROMETHANOPTERIN - COENZYME-M METHYLTRANSFERASE IN METHANOGENIC ARCHAEBACTERIA IS A MEMBRANE-PROTEIN
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DOI：
10.1007/bf00290817
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发表时间：
1992-08-01
影响因子：
2.8
通讯作者：
THAUER, RK
中科院分区：
文献类型：
--
作者：
FISCHER, R;GARTNER, P;THAUER, RK
描述了一种测定法，其允许直接测量产甲烷古细菌中催化甲基从N5-甲基四氢甲烷蝶呤（CH 3-H 4 MPT）转移至辅酶M（H-S-CoM）的酶活性。用这种方法的拓扑结构，部分纯化，和甲醇和乙酸盐生长的甲烷八叠球菌barkeri和H-2/CO2生长的甲烷热自养甲烷杆菌中的甲基转移酶的催化性能进行了研究。酶的活性被认为是几乎完全与膜部分，并需要洗涤剂溶解。甲醇培养的M.详细研究了巴克里。膜级分表现出由CH 3-H4 MPT（表观K（m）= 50 μ-M）和H-S-CoM（表观K（m）= 250 μ-M）形成约0.6-mu-mol.min-1.mg蛋白-1的CH 3-S-CoM的比活性。对于活性，需要催化量的柠檬酸钛（III）（表观K（m）= 15 μ M）和ATP（表观K（m）= 30 μ M）的存在。Ti（III）可以被还原的铁氧还蛋白取代。ATP不能被AMP、CTP、GTP、S-腺苷甲硫氨酸或ATP类似物取代。在不存在H-S-CoM的情况下，通过CH 3-H4 MPT将膜级分甲基化。这种甲基化依赖于Ti（III）和ATP。在不存在ATP和Ti（III）的情况下，甲基化的膜部分催化从CH 3-H4 MPT到H-S-CoM的甲基转移。在H-S-CoM存在下的脱甲基化也不需要Ti（III）或ATP。基于这些发现的甲基转移反应和酶的活化的机制提出。
An assay is described that allows the direct measurement of the enzyme activity catalyzing the transfer of the methyl group from N5-methyltetrahydromethanopterin (CH3-H4MPT) to coenzyme M (H-S-CoM) in methanogenic archaebacteria. With this method the topology, the partial purification, and the catalytic properties of the methyltransferase in methanol- and acetate-grown Methanosarcina barkeri and in H-2/CO2-grown Methanobacterium thermoautotrophicum were studied. The enzyme activity was found to be associated almost completely with the membrane fraction and to require detergents for solubilization. The transferase activity in methanol-grown M. barkeri was studied in detail. The membrane fraction exhibited a specific activity of CH3-S-CoM formation from CH3-H4MPT (apparent K(m) = 50-mu-M) and H-S-CoM (apparent K(m) = 250-mu-M) of approximately 0.6-mu-mol.min-1.mg protein-1. For activity the presence of Ti(III) citrate (apparent K(m) = 15-mu-M) and of ATP (apparent K(m) = 30-mu-M) were required in catalytic amounts. Ti(III) could be substituted by reduced ferredoxin. ATP could not be substituted by AMP, CTP, GTP, S-adenosylmethionine, or by ATP analogues. The membrane fraction was methylated by CH3-H4MPT in the absence of H-S-CoM. This methylation was dependent on Ti(III) and ATP. The methylated membrane fraction catalyzed the methyltransfer from CH3-H4MPT to H-S-CoM in the absence of ATP and Ti(III). Demethylation in the presence of H-S-CoM also did not require Ti(III) or ATP. Based on these findings a mechanism for the methyltransfer reaction and for the activation of the enzyme is proposed.