Kinetics and mechanism of acetyl-CoA synthase : a CO-channeling enzyme /
Acetyl-CoA synthase (ACS) is a bifunctional enzyme that catalyzes the reversible reduction of CO₂ to CO and the synthesis of acetyl-CoA from CO, coenzyme A, and a methyl group, at spatially distinct active sites (the C- and A-cluster, respectively). CO₂ was found to be a classical substrate for the...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2001.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=725912071&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Acetyl-CoA synthase (ACS) is a bifunctional enzyme that catalyzes the reversible reduction of CO₂ to CO and the synthesis of acetyl-CoA from CO, coenzyme A, and a methyl group, at spatially distinct active sites (the C- and A-cluster, respectively). CO₂ was found to be a classical substrate for the synthesis of acetyl-CoA. Being a rapid and tight binder of CO, hemoglobin (Hb) was employed as a "CO sponge" in an attempt to shut down acetyl-CoA synthesis from CO₂. Hb was unable to bind the intermediate CO and the rate of acetyl-CoA synthesis was not affected. This suggests that ACS tunneled CO through its interior via a solvent-inaccessible protein-encapsulated tunnel to the A-cluster where it was used to make acetyl-CoA. Steady-state initial rates of acetyl-CoA synthesis catalyzed by ACS were determined at various partial pressures of CO and CO₂. CO or CO₂ and a reductant activated ACS. CO and CO₂ were found to bind competitively to ACS (most likely at the C-cluster). At higher [CO] (> 100 ưM), ACS became inhibited. The mode of CO inhibition showed positive cooperativity and was uncompetitive with respect to CO₂. Roughly 10% activity remained at saturating CO pressures, indicating either a heterogeneous enzyme population or the diminished activity of a CO-bound enzyme form. During acetyl-CoA synthesis, CO is formed at the reductase site within ACS and is channeled more than 10 [Å] to the synthase site. By simultaneously measuring CO and reductant levels in real time, we have gained substantial insight into the mechanism of active-site coupling. Our studies reveal that the ACS active sites are coupled through the binding of a methylated corrinoid protein and coenzyme A to the synthase active site. This coupling altered the kinetic properties of the reductase site, re-routed CO to the A-cluster via the tunnel, and activated an unidentified redox reaction. These studies raise the possibility that kinetic order in enzyme mechanisms may be a general feature required for the synchronization of active sites in all multifunctional metabolite-channeling enzymes. |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xiii, 115 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 101-105). |