The oligomerization of CPS from E. coli was investigated in order to examine the influence of this property on the catalytic activity. Mutations at the two interfacial sites of oligomerization were constructed in an attempt to elucidate the mechanism for assembly of the (αβ)4 tetramer through disruption of the molecular binding interactions between monomeric units. The results are consistent with a model for the structure of the (αβ)2 dimer that is formed through molecular contact between two pairs of allosteric domains. No significant dependence of the specific catalytic activity on the protein concentration could be detected. The molecular tunnel within CPS was inspected in order to characterize the role on kinetic properties. Gln-22, Ala-23, and Gly-575 from the large subunit of CPS were substituted by mutagenesis with bulkier amino acids in an attempt to obstruct and/or hinder the passage of the unstable intermediate through the carbamate tunnel. The kinetic data are consistent with a model for the catalytic mechanism of CPS that requires the diffusion of carbamate through the interior of the enzyme from the site of synthesis within the N-terminal domain of the large subunit to the site of phosphorylation within the C-terminal domain to yield a final product carbamoyl phosphate. In addition, a unique feature of the carbamate tunnel has been noted where five highly conserved glutamates are located on a particular interior face of the tunnel. It has been postulated that the negative charge stabilizes the acid-labile intermediate, and facilitates catalysis. Also, the proposed gate keeping residues, Arg-306 and Arg-848, have been mutated to alanines to test their roles. However, since the arginines directly interact with MgATP, the mutation appeared to interrupt the binding of the substrate. The ammonia tunnel has been engineered to contain a hole to further support the proposed role of the tunnel that it is utilized in guiding diffusion of ammonia from the site of glutamine hydrolysis to the subsequent active site in the large subunit. Triple mutant αP360A/αH361A/βR265A exhibited kinetic behaviors consistent with a model of an impaired channeling.
| Identifer | oai:union.ndltd.org:TEXASAandM/oai:repository.tamu.edu:1969.1/332 |
| Date | 30 September 2004 |
| Creators | Kim, Jungwook |
| Contributors | Raushel, Frank M., Lindahl, Paul A., DeRose, Victoria J., Reinhart, Gregory D. |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Electronic Dissertation, text |
| Format | 2786562 bytes, 182625 bytes, electronic, application/pdf, text/plain, born digital |
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