The mutagenesis studies of differentially conserved residues within the carboxy phosphate and carbamate domains of Escherichia coli carbamoyl phosphate synthetase /
The role of individually conserved amino acids located in the amino- and carboxyl-terminal domains of CPS in catalysis and substrate binding were examined using site-directed mutagenesis studies as the primary tool. On the basis of amino acid sequence alignments and the X-ray crystal structure coor...
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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=737704141&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | The role of individually conserved amino acids located in the amino- and carboxyl-terminal domains of CPS in catalysis and substrate binding were examined using site-directed mutagenesis studies as the primary tool. On the basis of amino acid sequence alignments and the X-ray crystal structure coordinates for biotin carboxylase and D-ala-D-ala ligase, a working model for the three-dimensional fold of the carbamate domain of CPS was constructed. This model was used to select 10 residues within the carbamate domain of CPS for modification and subsequent characterization of the . kinetic constants for the mutant proteins. Residues R571, R675, R715, D753, E761, N827, Q829 were changed to alanine residues; residues E841 and Q845 to glutamine; and residue N843 to aspartate. The mutants E761A, E841Q, N843D, and R845Q were diminished in their ability to synthesize carbamoyl phosphate. The R715A, Q829A, and R675A mutants displayed elevated Michaelis constants for MGADP in the partial back reaction. The mutants E761A, N827A, E841A, N843D, and R845Q showed significant increases in the Michaelis constants for either bicarbonate or carbamoyl phosphate. No significant alterations were noted upon mutation of either R571 or D753 to an alanine residue and thus these amino acids appear to be nonessential for binding and/or catalytic activity. To probe the differences in catalytic functions between the carboxy phosphate and carbarnate domains of CPS, a chimeric protein was constructed where the carboxylterminal half of the protein was duplicated. This protein was not expressed. These results suggest that the differences in sequences of the two homologous halves are essential for activity and stability of used as a guide for selection of six target residues at positions E783, S789, K891, E892, D904, and E910 in order to probe the mechanism for the catalytic regulation by omithine. No significant changes in the allosteric activation by omithine were observed upon mutation of either D904 or E91 0, and thus these residues appear to be nonessential for allosteric regulation of this ligand. These results also indicated that the hydrogen bonding interaction between the side chain of K891 and the carbonyl oxygen back bone of S789 is important in the regulatory properties of omithine. More significantly, these results also determined the involvement of the side chain amino group of omithine in neutralization of the negatively charged pocket created by the carboxylate groups of two glutainate residues at positions 783 and 892, which seem to be essential for enzymatic activation by omithine. |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xii, 146 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references: pages 142-145. |