PURIFICATION AND PROPERTIES OF N(5)-METHYLTETRAHYDROMETHANOPTERIN COENZYME-M METHYLTRANSFERASE FROM METHANOBACTERIUM-THERMOAUTOTROPHICUM

被引:71
作者
GARTNER, P
ECKER, A
FISCHER, R
LINDER, D
FUCHS, G
THAUER, RK
机构
[1] MAX PLANCK INST TERR MIKROBIOL,MARBURG,GERMANY
[2] UNIV ULM,ANGEW MIKROBIOL ABT,W-7900 ULM,GERMANY
[3] UNIV GIESSEN,FACHBEREICH HUMANMED,INST BIOCHEM,W-6300 GIESSEN,GERMANY
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1993年 / 213卷 / 01期
关键词
D O I
10.1111/j.1432-1033.1993.tb17792.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-hydroxy-benzimidazolyl 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 V(max)) of the purified methyltransferase preparation was 11.6 mumol . min-1 . mg protein-1. The apparent K(m) for N5-methyltetrahydromethanopterin was 260 muM and that for coenzyme M was 60 muM. The preparation was absolutely dependent on the presence of Ti(III) for activity. ATP enhanced the activity 1.5-2-fold.
引用
收藏
页码:537 / 545
页数:9
相关论文
共 38 条
[1]   PARTICIPATION OF COB(I)ALAMIN IN THE REACTION CATALYZED BY METHIONINE SYNTHASE FROM ESCHERICHIA-COLI - A STEADY-STATE AND RAPID REACTION KINETIC-ANALYSIS [J].
BANERJEE, RV ;
FRASCA, V ;
BALLOU, DP ;
MATTHEWS, RG .
BIOCHEMISTRY, 1990, 29 (50) :11101-11109
[2]  
BANERJEE RV, 1989, J BIOL CHEM, V264, P13888
[3]   MECHANISM OF REDUCTIVE ACTIVATION OF COBALAMIN-DEPENDENT METHIONINE SYNTHASE - AN ELECTRON-PARAMAGNETIC RESONANCE SPECTROELECTROCHEMICAL STUDY [J].
BANERJEE, RV ;
HARDER, SR ;
RAGSDALE, SW ;
MATTHEWS, RG .
BIOCHEMISTRY, 1990, 29 (05) :1129-1135
[4]  
BECHER B, 1992, FEMS MICROBIOL LETT, V91, P239
[5]   N-5-METHYL-TETRAHYDROMETHANOPTERIN - COENZYME-M METHYLTRANSFERASE OF METHANOSARCINA STRAIN GO1 IS AN NA+-TRANSLOCATING MEMBRANE-PROTEIN [J].
BECHER, B ;
MULLER, V ;
GOTTSCHALK, G .
JOURNAL OF BACTERIOLOGY, 1992, 174 (23) :7656-7660
[6]   ENERGETICS OF METHANOGENESIS STUDIED IN VESICULAR SYSTEMS [J].
BLAUT, M ;
MULLER, V ;
GOTTSCHALK, G .
JOURNAL OF BIOENERGETICS AND BIOMEMBRANES, 1992, 24 (06) :529-546
[7]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[8]   RELATEDNESS OF STRAINS DELTA-H AND MARBURG OF METHANOBACTERIUM-THERMOAUTOTROPHICUM [J].
BRANDIS, A ;
THAUER, RK ;
STETTER, KO .
ZENTRALBLATT FUR BAKTERIOLOGIE MIKROBIOLOGIE UND HYGIENE I ABTEILUNG ORIGINALE C-ALLGEMEINE ANGEWANDTE UND OKOLOGISCHE MIKROBIOLOGIE, 1981, 2 (04) :311-317
[9]   SALT DEPENDENCE, KINETIC-PROPERTIES AND CATALYTIC MECHANISM OF N-FORMYLMETHANOFURAN - TETRAHYDROMETHANOPTERIN FORMYLTRANSFERASE FROM THE EXTREME THERMOPHILE METHANOPYRUS-KANDLERI [J].
BREITUNG, J ;
BORNER, G ;
SCHOLZ, S ;
LINDER, D ;
STETTER, KO ;
THAUER, RK .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1992, 210 (03) :971-981
[10]   SPECTROPHOTOMETRIC DETERMINATION OF HYDROGEN SULFIDE IN NATURAL WATERS [J].
CLINE, JD .
LIMNOLOGY AND OCEANOGRAPHY, 1969, 14 (03) :454-&