Mechanistic studies of argininosuccinate lyase, adenylosuccinate lyase, glycogen synthetase, lactose synthetase, and phosphoglucomutase /
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| Other Authors: | , , |
| Format: | Thesis Book |
| Language: | English |
| Published: |
1988.
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| Subjects: | |
| Online Access: | ProQuest, Abstract Link to OAKTrust copy |
| Abstract: | A new method for the determination of dissociation rates of enzyme-substrate complexes has been developed. The rate of exchange of a labeled product back into the substrate is measured during catalysis of the forward reaction when the forward reaction is kept far from equilibrium by the enzymatic removal of the nonexchanging product. The ratio of the exchange rate and the rate for product formation is then determined at various concentrations of the exchanging product. A plot of this ratio is a diagnostic indication of the kinetic mechanism and the relative rates of product dissociation from the binary and ternary enzyme complexes. The mechanism of the argininosuccinate lyase reaction has been probed by the measurement of the effects of isotope substitution at the reaction centers. Data have been interpreted to suggest that argininosuccinate lyase catalyzes the cleavage of argininosuccinate via a carbanion intermediate. The proton abstraction step is not rate limiting, but the protonation of the guanidino group and carbon-nitrogen bond cleavage are kinetically significant. Monofluorofumarate was tested as an alternative substrate and inhibitor for adenylosuccinate lyase. Monofluorofumarate was found to be a slow substrate. The initial reaction product when monofluorofumarate was incubated with AMP in the presence of adenylosuccinate lyase has been determined to be 2-fluoro-adenylosuccinate. This molecule lost HF spontaneously, and the subsquent intermediate was rapidly hydrolyzed to oxalacetate and AMP. A similar reaction scheme was also observed when AICAR was utilized. The reaction mechanisms for glycogen synthetase and lactose synthetase were examined. Large α-secondary isotope effect on V[max] have been interpreted to show that an oxocarbonium ion is an intermediate in this reaction mechanism. Nojirimycin-6-phosphate was tested as a substrate and inhibitor for phosphoglucomutase. In the absence of glucose-1,6-bisphosphate, the incubation of phosphoglucomutase with no jirimycin-6-phophate resulted in complete inactivation of all enzyme activity. It has been shown that the phosphorylated form of phosphoglucomutase catalyzes the phosphorylation of nojirimycin-6-phosphate at C-1... |
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| Item Description: | Typescript (photocopy). Vita. "Major subject: Chemistry." |
| Physical Description: | xi, 119 leaves : illustrations ; 29 cm |
| Bibliography: | Includes bibliographical references (leaves 112-118). |