The nature of the coenzyme A binding site of carbon monoxide dehydrogenase from Clostridium thermoaceticum /
Carbon monoxide dehydrogenase from Clostridium thermoaceticum catalyzes the synthesis of acetyl-coenzyme A (acetyl-CoA) from CO, CoA and a methylated corrinoid-iron-sulfur protein. The enzyme is an [ ] 2[ beta]2 tetramer with a novel Ni-X- Fe4S, A-cluster active site located in each cc subunit. CO...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1998.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=737703841&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Carbon monoxide dehydrogenase from Clostridium thermoaceticum catalyzes the synthesis of acetyl-coenzyme A (acetyl-CoA) from CO, CoA and a methylated corrinoid-iron-sulfur protein. The enzyme is an [ ] 2[ beta]2 tetramer with a novel Ni-X- Fe4S, A-cluster active site located in each cc subunit. CO and the methyl group appear to bind the Ni of the A-cluster, while the site of CoA binding remains unknown. To characterize the CoA binding site, binding parameters for CoA were determined by equilibrium dialysis. CoA bound to native enzyme with Kd= 10 ︢8 uM and n = 0.2 ︢0.1 moles per cop dimer. For reasons not understood, only -30% of the enzyme's (xp dimeric units are functional, and only these contain A-clusters with labile Ni ions. The n value obtained for CoA binding suggests that CoA binds only to (xp units with functional A-clusters. The CoA binding properties of enzyme lacking the labile Ni were not significantly different, indicating that CoA does not bind the labile Ni of the A-cluster. However, mechanistic considerations appear to require that the thiolate portion of CoA binds near the Ni of the A-cluster. That CoA does not bind the labile Ni is further evidenced by the observation that up to 200 uM CoA does not inhibit the removal of the labile Ni by phen. The free energy of binding could correspond to the formation of a strong hydrogen bond between the CoA thiol and a base in the active site, which activates the sulfur towards nucleophilic attack. Additional equilibrium dialysis experiments indicate that CoA does not bind CO-reduced enzyme. Moreover, the NiFeC EPR signal exhibited by the A red-CO state was unaffected by incubating CO-reduced enzyme with up to 200 @M CoA. These results suggest a COdependent conformational change that prevents binding of CoA. They also provide further evidence that the A red-CO state is not a catalytic intermediate. The binding of CoA to CODH reduced with titanium (III) citrate (Kd = 3 6 ︢16 uM, n = 0. 16 ︢0.08) is not significantly different than the binding parameters of native enzyme, indicating that the D-site is not involved in CoA binding (Barondeau, D. P.; Lindahl, P. A. (1997) J Am. Chem. Soc. 119, 3959-3970). |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xi, 73 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references: pages 48-51. |