Properties of the two isoenzymes of methyl‐coenzyme M reductase in Methanobacterium thermoautotrophicum

Methyl‐coenzyme M reductase (MCR) catalyses the methane‐forming step in the energy metabolism of methanogenic Archaea. It brings about the reduction of methyl‐coenzyme M (CH3‐S‐CoM) by 7‐mercaptoheptanoylthreonine phosphate (H‐S‐HTP). Methanobacterium thermoautotrophicum contains two isoenzymes of M...

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Veröffentlicht in:European journal of biochemistry 1993-10, Vol.217 (2), p.587-595
Hauptverfasser: BONACKER, Lutz G., BAUDNER, Siegfried, MÖRSCHEL, Erhard, BÖCHER, Reinhard, THAUER, Rudolf K.
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container_issue 2
container_start_page 587
container_title European journal of biochemistry
container_volume 217
creator BONACKER, Lutz G.
BAUDNER, Siegfried
MÖRSCHEL, Erhard
BÖCHER, Reinhard
THAUER, Rudolf K.
description Methyl‐coenzyme M reductase (MCR) catalyses the methane‐forming step in the energy metabolism of methanogenic Archaea. It brings about the reduction of methyl‐coenzyme M (CH3‐S‐CoM) by 7‐mercaptoheptanoylthreonine phosphate (H‐S‐HTP). Methanobacterium thermoautotrophicum contains two isoenzymes of MCR, designated MCR I and MCR II, which are expressed differentially under different conditions of growth. These two isoenzymes have been separated, purified and their catalytic and spectroscopic properties determined. Initial‐velocity measurements of the two‐substrate reaction showed that the kinetic mechanism for both isoenzymes involved ternary‐complex formation. Double reciprocal plots of initial rates versus the concentration of either one of the two substrates at different constant concentrations of the other substrate were linear and intersected on the abcissa to the left of the 1/v axis. The two purified isoenzymes differed in their Km values for H‐S‐HTP and for CH3‐S‐CoM and in Vmax. MCR I displayed a Km for H‐S‐HTP of 0.1–0.3 mM, a Km for CH3 S‐CoM of 0.6–0.8 mM and a Vmax of about 6 μmol · min−1· mg−1 (most active preparation). MCR II showed a Km for H‐S‐HTP of 0.4–0.6 mM, a Km for CH3‐S‐CoM of 1.3–1.5 mM and a Vmax of about 21 μmol · min−1· mg−1 (most active preparation). The pH optimum of MCR I was 7.0–7.5 and that of MCR II 7.5–8.0. Both isoenzymes exhibited very similar temperature activity optima and EPR properties. The location of MCR I and of MCR II within the cell, determined via immunogold labeling, was found to be essentially identical. The possible basis for the existance of MCR isoenzymes in M. thermoautotrophicum is discussed.
doi_str_mv 10.1111/j.1432-1033.1993.tb18281.x
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It brings about the reduction of methyl‐coenzyme M (CH3‐S‐CoM) by 7‐mercaptoheptanoylthreonine phosphate (H‐S‐HTP). Methanobacterium thermoautotrophicum contains two isoenzymes of MCR, designated MCR I and MCR II, which are expressed differentially under different conditions of growth. These two isoenzymes have been separated, purified and their catalytic and spectroscopic properties determined. Initial‐velocity measurements of the two‐substrate reaction showed that the kinetic mechanism for both isoenzymes involved ternary‐complex formation. Double reciprocal plots of initial rates versus the concentration of either one of the two substrates at different constant concentrations of the other substrate were linear and intersected on the abcissa to the left of the 1/v axis. The two purified isoenzymes differed in their Km values for H‐S‐HTP and for CH3‐S‐CoM and in Vmax. MCR I displayed a Km for H‐S‐HTP of 0.1–0.3 mM, a Km for CH3 S‐CoM of 0.6–0.8 mM and a Vmax of about 6 μmol · min−1· mg−1 (most active preparation). MCR II showed a Km for H‐S‐HTP of 0.4–0.6 mM, a Km for CH3‐S‐CoM of 1.3–1.5 mM and a Vmax of about 21 μmol · min−1· mg−1 (most active preparation). The pH optimum of MCR I was 7.0–7.5 and that of MCR II 7.5–8.0. Both isoenzymes exhibited very similar temperature activity optima and EPR properties. The location of MCR I and of MCR II within the cell, determined via immunogold labeling, was found to be essentially identical. 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It brings about the reduction of methyl‐coenzyme M (CH3‐S‐CoM) by 7‐mercaptoheptanoylthreonine phosphate (H‐S‐HTP). Methanobacterium thermoautotrophicum contains two isoenzymes of MCR, designated MCR I and MCR II, which are expressed differentially under different conditions of growth. These two isoenzymes have been separated, purified and their catalytic and spectroscopic properties determined. Initial‐velocity measurements of the two‐substrate reaction showed that the kinetic mechanism for both isoenzymes involved ternary‐complex formation. Double reciprocal plots of initial rates versus the concentration of either one of the two substrates at different constant concentrations of the other substrate were linear and intersected on the abcissa to the left of the 1/v axis. The two purified isoenzymes differed in their Km values for H‐S‐HTP and for CH3‐S‐CoM and in Vmax. MCR I displayed a Km for H‐S‐HTP of 0.1–0.3 mM, a Km for CH3 S‐CoM of 0.6–0.8 mM and a Vmax of about 6 μmol · min−1· mg−1 (most active preparation). MCR II showed a Km for H‐S‐HTP of 0.4–0.6 mM, a Km for CH3‐S‐CoM of 1.3–1.5 mM and a Vmax of about 21 μmol · min−1· mg−1 (most active preparation). The pH optimum of MCR I was 7.0–7.5 and that of MCR II 7.5–8.0. Both isoenzymes exhibited very similar temperature activity optima and EPR properties. The location of MCR I and of MCR II within the cell, determined via immunogold labeling, was found to be essentially identical. The possible basis for the existance of MCR isoenzymes in M. thermoautotrophicum is discussed.</description><subject>Bacteriology</subject><subject>Biological and medical sciences</subject><subject>Catalysis</subject><subject>Electron Spin Resonance Spectroscopy</subject><subject>Fundamental and applied biological sciences. 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Enzymes</subject><subject>Methane - metabolism</subject><subject>Methanobacterium - enzymology</subject><subject>Methanobacterium - growth &amp; development</subject><subject>Methanobacterium thermoautotrophicum</subject><subject>Microbiology</subject><subject>Oxidation-Reduction</subject><subject>Oxidoreductases - chemistry</subject><subject>Oxidoreductases - isolation &amp; purification</subject><subject>Oxidoreductases - metabolism</subject><subject>Phosphothreonine - analogs &amp; derivatives</subject><subject>Phosphothreonine - metabolism</subject><subject>Spectrophotometry, Ultraviolet</subject><issn>0014-2956</issn><issn>1432-1033</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1993</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqVkd-K1DAUxoMo6zjrIwhFxLt2869J6o3osv9gF4XV65Cmp0yGthmTlN3xykfwGX0SW6bM7bKBEDjf73wnnA-h9wQXZDpn24JwRnOCGStIVbEi1URRRYrHF2h1lF6iFcaE57QqxWv0JsYtxlhUQp6gE0UpE5iukPse_A5CchAz32ZpA1l68JmLHobf-_5Q7SFt9t2_P3_tUs3usgDNaJOJkLkhu5sAM_ja2ATBjf3sE3pvxuTT5L9xduxP0avWdBHeLu8a_by8-HF-nd9-u7o5_3KbW06IzIWtACsgYDDldYMFNq1oVSUo47WtKOcCyxIItZwqKWmjGlkShRlXpWFNxdbo48F3F_yvEWLSvYsWus4M4MeopcCMlEQ8CRKhGJflDH46gDb4GAO0ehdcb8JeE6znQPRWz1vX89b1HIheAtGPU_O7ZcpY99AcW5cEJv3DoptoTdcGM1gXjxiTkpHprtHnA_bgOtg_4wP68uLrfakk-w_BoKnd</recordid><startdate>199310</startdate><enddate>199310</enddate><creator>BONACKER, Lutz G.</creator><creator>BAUDNER, Siegfried</creator><creator>MÖRSCHEL, Erhard</creator><creator>BÖCHER, Reinhard</creator><creator>THAUER, Rudolf K.</creator><general>Blackwell Publishing Ltd</general><general>Blackwell</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>C1K</scope><scope>7X8</scope></search><sort><creationdate>199310</creationdate><title>Properties of the two isoenzymes of methyl‐coenzyme M reductase in Methanobacterium thermoautotrophicum</title><author>BONACKER, Lutz G. ; BAUDNER, Siegfried ; MÖRSCHEL, Erhard ; BÖCHER, Reinhard ; THAUER, Rudolf K.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4117-6c9e08e1ea024bd060af6f896234bc92446075e12c428772d8d751803485a3d93</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1993</creationdate><topic>Bacteriology</topic><topic>Biological and medical sciences</topic><topic>Catalysis</topic><topic>Electron Spin Resonance Spectroscopy</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Immunohistochemistry</topic><topic>Isoenzymes - chemistry</topic><topic>Isoenzymes - isolation &amp; purification</topic><topic>Isoenzymes - metabolism</topic><topic>Kinetics</topic><topic>Mesna - analogs &amp; derivatives</topic><topic>Mesna - metabolism</topic><topic>Metabolism. Enzymes</topic><topic>Methane - metabolism</topic><topic>Methanobacterium - enzymology</topic><topic>Methanobacterium - growth &amp; development</topic><topic>Methanobacterium thermoautotrophicum</topic><topic>Microbiology</topic><topic>Oxidation-Reduction</topic><topic>Oxidoreductases - chemistry</topic><topic>Oxidoreductases - isolation &amp; purification</topic><topic>Oxidoreductases - metabolism</topic><topic>Phosphothreonine - analogs &amp; derivatives</topic><topic>Phosphothreonine - metabolism</topic><topic>Spectrophotometry, Ultraviolet</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>BONACKER, Lutz G.</creatorcontrib><creatorcontrib>BAUDNER, Siegfried</creatorcontrib><creatorcontrib>MÖRSCHEL, Erhard</creatorcontrib><creatorcontrib>BÖCHER, Reinhard</creatorcontrib><creatorcontrib>THAUER, Rudolf K.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>European journal of biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>BONACKER, Lutz G.</au><au>BAUDNER, Siegfried</au><au>MÖRSCHEL, Erhard</au><au>BÖCHER, Reinhard</au><au>THAUER, Rudolf K.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Properties of the two isoenzymes of methyl‐coenzyme M reductase in Methanobacterium thermoautotrophicum</atitle><jtitle>European journal of biochemistry</jtitle><addtitle>Eur J Biochem</addtitle><date>1993-10</date><risdate>1993</risdate><volume>217</volume><issue>2</issue><spage>587</spage><epage>595</epage><pages>587-595</pages><issn>0014-2956</issn><eissn>1432-1033</eissn><coden>EJBCAI</coden><abstract>Methyl‐coenzyme M reductase (MCR) catalyses the methane‐forming step in the energy metabolism of methanogenic Archaea. It brings about the reduction of methyl‐coenzyme M (CH3‐S‐CoM) by 7‐mercaptoheptanoylthreonine phosphate (H‐S‐HTP). Methanobacterium thermoautotrophicum contains two isoenzymes of MCR, designated MCR I and MCR II, which are expressed differentially under different conditions of growth. These two isoenzymes have been separated, purified and their catalytic and spectroscopic properties determined. Initial‐velocity measurements of the two‐substrate reaction showed that the kinetic mechanism for both isoenzymes involved ternary‐complex formation. Double reciprocal plots of initial rates versus the concentration of either one of the two substrates at different constant concentrations of the other substrate were linear and intersected on the abcissa to the left of the 1/v axis. The two purified isoenzymes differed in their Km values for H‐S‐HTP and for CH3‐S‐CoM and in Vmax. MCR I displayed a Km for H‐S‐HTP of 0.1–0.3 mM, a Km for CH3 S‐CoM of 0.6–0.8 mM and a Vmax of about 6 μmol · min−1· mg−1 (most active preparation). MCR II showed a Km for H‐S‐HTP of 0.4–0.6 mM, a Km for CH3‐S‐CoM of 1.3–1.5 mM and a Vmax of about 21 μmol · min−1· mg−1 (most active preparation). The pH optimum of MCR I was 7.0–7.5 and that of MCR II 7.5–8.0. Both isoenzymes exhibited very similar temperature activity optima and EPR properties. The location of MCR I and of MCR II within the cell, determined via immunogold labeling, was found to be essentially identical. The possible basis for the existance of MCR isoenzymes in M. thermoautotrophicum is discussed.</abstract><cop>Oxford, UK</cop><pub>Blackwell Publishing Ltd</pub><pmid>8223602</pmid><doi>10.1111/j.1432-1033.1993.tb18281.x</doi><tpages>9</tpages></addata></record>
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subjects Bacteriology
Biological and medical sciences
Catalysis
Electron Spin Resonance Spectroscopy
Fundamental and applied biological sciences. Psychology
Immunohistochemistry
Isoenzymes - chemistry
Isoenzymes - isolation & purification
Isoenzymes - metabolism
Kinetics
Mesna - analogs & derivatives
Mesna - metabolism
Metabolism. Enzymes
Methane - metabolism
Methanobacterium - enzymology
Methanobacterium - growth & development
Methanobacterium thermoautotrophicum
Microbiology
Oxidation-Reduction
Oxidoreductases - chemistry
Oxidoreductases - isolation & purification
Oxidoreductases - metabolism
Phosphothreonine - analogs & derivatives
Phosphothreonine - metabolism
Spectrophotometry, Ultraviolet
title Properties of the two isoenzymes of methyl‐coenzyme M reductase in Methanobacterium thermoautotrophicum
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