Structural and functional domain study of Escherichia coli carbamoyl phosphate synthetase by site-directed mutagenesis /
Site-directed mutagenesis has been used as a primary tool to study the functional and structural properties of Escherichia coli carbamoyl phosphate synthetase. Conserved glycines found in the subunit interaction domain of both the N-tertninal and C-terminal half of CPS were individually changed to...
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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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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=743266741&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Site-directed mutagenesis has been used as a primary tool to study the functional and structural properties of Escherichia coli carbamoyl phosphate synthetase. Conserved glycines found in the subunit interaction domain of both the N-tertninal and C-terminal half of CPS were individually changed to isoleucines. Mutations in the N-tertninal half elevated the K. for MGATP in the bicarbonate-dependent ATP hydrolysis reaction as well as the K. for bicarbonate. Mutants G211 and G91I affected the mechanism for carbamoyl phosphate production. The C-terininal mutations affected the K. for MGADP in the ATP synthesis reaction. Conserved glycines in the allosteric domain were individually changed to alanines, valines and isoleucines. The arosteric effect exerted by omithine was shown to diminish as the glycine at position 968 is progressively changed to isoleucine. The aflosteric effect of the inactivator UMP is shut off when the glycine at position 968 is changed to an alanine. To further investigate this region conserved non-glycine residues were individually changed to alanines. The T977A mutant shut off the inactivating effect of UMP while none of the non-glycine mutants had any appreciable affect on the activator omithine. To investigate the function of the "unknown" region conserved glycines were changed to isoleucines and conserved charged polar amino acids were changed to alanines. The G5681 mutant uncoupled the enzyme, which resulted in very little carbamoyl phosphate produced. This mutation also showed a synergetic effect in the hydrolysis of ATP as the concentration of ATP was increased. This result suggest that the two ATP sites in the N-ten-ninal and C-terininal halves are believed to be acting independently of each other phosphorylating bicarbonate. The R435A mutation showed similar synergetic hydrolysis of ATP as the mononucleotides concentration was increased. This mutation showed no activating effect with glutamine but ammonia was still able to activate the enzyme. However, the R435A mutant produced carbamoyl phosphate at a rate that was 40 % the rate of ATP hydrolysis when ammonia or glutamine was present. |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xi, 116 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references. |