Gärtner P, Ecker A, Fischer R, Linder D, Fuchs G, Thauer R K
Laboratorium für Mikrobiologie des Fachbereichs Biologie, Philipps-Universität Marburg, Germany.
Eur J Biochem. 1993 Apr 1;213(1):537-45. doi: 10.1111/j.1432-1033.1993.tb17792.x.
N5-Methyltetrahydromethanopterin:coenzyme M meth-yltransferase is an integral membrane protein found in methanogenic archaea. It catalyzes an energy-conserving step in methane formation from CO2 and from acetate. The enzyme from Methanobacterium thermoautotrophicum (strain Marburg) has been purified 30-fold to apparent homogeneity. The purified enzyme had an apparent molecular mass of 670 kDa and was composed of seven different polypeptides of 34 kDa, 28 kDa, 24 kDa, 23 kDa, 21 kDa, 13 kDa, and 12 kDa. The N-terminal amino acid sequences of these polypeptides were determined. The native 670-kDa enzyme was found to contain 7.6 mol 5-hydroxybenzimidazolyl cobamide/mol, 37 mol non-heme iron/mol and 34 mol acid-labile sulfur/mol. Cobalt analyses after sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that the corrinoid was bound to the 23-kDa polypeptide. The apparent molecular masses of the polypeptides given above were determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis without boiling the samples prior to analysis. When the samples were boiled, as is usually done, the 23-kDa polypeptide changed its apparent molecular mass to 33 kDa and the 21-kDa, 24-kDa, and 28-kDa polypeptides formed aggregates. The specific activity (apparent Vmax) of the purified methyltransferase preparation was 11.6 mumol.min-1.mg protein-1. The apparent Km for N5-methyltetrahydromethanopterin was 260 microM and that for coenzyme M was 60 microM. The preparation was absolutely dependent on the presence of Ti(III) for activity. ATP enhanced the activity 1.5-2-fold.
N5-甲基四氢甲蝶呤：辅酶M甲基转移酶是一种存在于产甲烷古菌中的整合膜蛋白。它催化从二氧化碳和乙酸盐形成甲烷过程中的一个能量守恒步骤。来自嗜热自养甲烷杆菌（马尔堡菌株）的这种酶已被纯化30倍，达到表观均一性。纯化后的酶表观分子量为670 kDa，由7种不同的多肽组成，分子量分别为34 kDa、28 kDa、24 kDa、23 kDa、21 kDa、13 kDa和12 kDa。测定了这些多肽的N端氨基酸序列。发现天然的670-kDa酶每摩尔含有7.6摩尔5-羟基苯并咪唑基钴胺素、37摩尔非血红素铁和34摩尔酸不稳定硫。十二烷基硫酸钠/聚丙烯酰胺凝胶电泳后的钴分析表明，类咕啉与23-kDa多肽结合。上述多肽的表观分子量是通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳测定的，分析前未对样品进行煮沸处理。当像通常那样对样品进行煮沸时，23-kDa多肽的表观分子量变为33 kDa，21-kDa、24-kDa和28-kDa多肽形成聚集体。纯化的甲基转移酶制剂的比活性（表观Vmax）为11.6 μmol·min-1·mg蛋白-1。N5-甲基四氢甲蝶呤的表观Km为260 μM，辅酶M的表观Km为60 μM。该制剂的活性绝对依赖于Ti(III)的存在。ATP使活性提高1.5至2倍。
