Methylation of carbon monoxide dehydrogenase and the mechanism of acetyl-CoA synthesis /
Carbon monoxide dehydrogenase from Clostridium thermoaceticum catalyzes a unique bioorganometallic reaction; the synthesis of acetyl-coenzyme A from CO, CoA, and a methyl cation transferred to the enzyme from a corrinoid-iron-sulfur protein. The reaction is catalyzed at the A-cluster, composed of a...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1996.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=739640991&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Carbon monoxide dehydrogenase from Clostridium thermoaceticum catalyzes a unique bioorganometallic reaction; the synthesis of acetyl-coenzyme A from CO, CoA, and a methyl cation transferred to the enzyme from a corrinoid-iron-sulfur protein. The reaction is catalyzed at the A-cluster, composed of a labile Ni (removable by 1,10-phenanthroline) linked through an unidentified bridge to an Fe4S4 cluster. A procedure was developed to methylate CODH in a viable manner, so that all bound methyl groups could subsequently react with CO and COAS- (or OH-) to yield acetyl-CoA (or acetate). Methyl could not bind to enzyme lacking the labile Ni, but it could bind to such samples after incubation in aqueous Ni", a process known to reinsert the labile Ni and reactivate the enzyme. Moreover, bound methyl inhibited the ability of phen to remove the labile Ni, and the amount of methyl that could bind the enzyme approximately correlated with the amount of labile Ni. The reactivity pattern evident from these experiments provides strong evidence that the methyl used in acetyl-CoA synthesis binds at the labile Ni. In contrast to earlier studies in which the A-cluster itself was thought to be reductively activated for methylation (to an S = 1/2 CO- bound form), evidence is presented that the site reduced is not the A-cluster itself, but another species called D. Both methylated- and acetyl-intermediates appear to be EPR-silent. The D site appears to be an n = 2 redox agent that functions to reduce the oxidized A-cluster upon methylation, and to oxidize the A-cluster as the product acetyl-CoA dissociates. D is EPR-silent in both of its oxidation states, and is not any of the known metal clusters in the enzyme. D may be a special pair of cysteines coordinated to the labile Ni that can be oxidized to the disulfide form at unusually low potentials ([] -530 mV vs. NHE). Catalytic mechanisms that do not include D or its functional equivalent or that employ the reduced S = 1/2 CO-bound form of the A-cluster as an intermediate do not appear to be correct. A new catalytic mechanism incorporating the results of this study is proposed. |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xiii, 118 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references: pages 76-85. |