Mechanistic studies of glutaminase activity of a glutamine amidotransferase, carbamoyl phosphate synthetase from Escherichia coli /
| Main Author: | |
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| Other Authors: | , , |
| Format: | Thesis Book |
| Language: | English |
| Published: |
1991.
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| Subjects: | |
| Online Access: | Link to OAKTrust copy |
| Abstract: | The mechanistic properties of the glutaminase reaction of E. coli carbamoyl phosphate synthetase have been studied. Chemical modification study was carried out using diethyl pyrocarbonate (DEPC) to investigate the involvement of histidine residue in the hydrolysis of glutamine by the enzyme. The glutaminase activity of the holo enzyme was inactivated with a second-order rate constant of 7.1M^-1min^-1, and that of the isolated small subunit enzymes was inactivated with a constant of 0.19mM^-1min^-1 by the incubation with DEPC. The formation of N-carbethoxyhistidine in the difference spectra, and the data for the 14C -DEPC labeling studies are consistent with the modification of histidine residues by DEPC. These findings indicated that histidine residue is essential for the glutaminase activity of the enzyme. The pH and deuterium oxide isotope studies of the glutaminase reaction were carried out. .The values of pKa and Δ pKa in D2O of the isolated small subunit are consistent with the involvement of histidine in the hydrolysis of glutamine by the enzyme. The role of the four conserved histidine residues in the small subunit for the H353N mutant is unable to hydrolyze glutamine. However the data for 14C-glutamine labeling study showed that glutamine can bind to the H353N mutant. The H312N mutant shows a Michaelis constant for glutamine 50-fold larger than the wild-type value but the maximal rate of the glutamine hydrolysis in unchanged. These results are consistent with His-353 functioning as a general base/acid catalyst for proton transfers while His-312 serves a critical role for the binding of glutamine to the active site. The radioactive substrate labeling and the proton exchange in D2O experiments were done to investigate the putative thioester intermediate in the glutamine hydrolysis. It was demonstrated that the enzyme-bound 14C-glutamine decomposed as a function of time upon the denaturation of the protein in the wild-type but was washed out in the H353N mutant. The results indicate that the enzyme-glutamine complex formed in the wild-type enzyme is a covalent one. Oroton exchange was not detected by the NMR method. A mechanism for the hydrolysis of glutamine which is consistent with these results is proposed... |
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| Item Description: | Typescript (photocopy). Vita. "Major subject: Chemistry." |
| Physical Description: | xi, 120 leaves : illustrations ; 29 cm |
| Bibliography: | Includes bibliographical references. |