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PURIFICATION AND KINETIC STUDIES OF ASPARTASE FROM ESCHERICHIA COLI AND EFFECT OF S-2,3-DICARBOXY AZIRIDINE ON FUMARASE FROM PIG HEART

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Title: PURIFICATION AND KINETIC STUDIES OF ASPARTASE FROM ESCHERICHIA COLI AND EFFECT OF S-2,3-DICARBOXY AZIRIDINE ON FUMARASE FROM PIG HEART
Author: GREENHUT, JOAN
Degree: Doctor of Philosophy thesis
Abstract: Aspartase has been purified from E. coli B cells using high performance liquid ion exchange chromatography. This procedure allows rapid purification of the enzyme with >80% recovery of total activity. The kinetic mechanism of aspartase, determined from initial velocity experiments, is best described by the equilibrium ordered addition of a divalent metal cation to free enzyme followed by binding of aspartate. Following interconversion of the central complex, NH(,4)('+) and fumarate are randomly released. Michaelis constants for each substrate are: 85 mM for NH(,4)('+), 0.17 mM for fumarate, 0.6-1.0 mM for aspartate and 9.0 (mu)M for Mg('2+). From the results of inhibition studies using structural analogues of aspartate, it was determined that aspartase recognizes the (beta)-carboxylate group of aspartate. The (alpha)-amino group contributes very little to the binding of aspartate to the enzyme. Compounds having an -OH group in addition to the (beta)-carboxylate had no effect on the rate of deamination of aspartate, suggesting a restricted geometry at the active site. S-2,3-Dicarboxy aziridine was found to be a potent competitive inhibitor of aspartase (K(,i) = 0.1 (mu)M) and fumarase (K(,i) = 0.08 (mu)M). The aziridine did not inactivate either enzyme nor did it exhibit any observable substrate activity. It is likely that it functions as a transition state analogue mimicking the carbanion intermediate found in the normal catalytic reaction. The aziridine inhibited fumarate utilization in ruptured but not intact mitochondria.
Citation: GREENHUT, JOAN. (1985) "PURIFICATION AND KINETIC STUDIES OF ASPARTASE FROM ESCHERICHIA COLI AND EFFECT OF S-2,3-DICARBOXY AZIRIDINE ON FUMARASE FROM PIG HEART." Doctoral Thesis, Rice University. http://hdl.handle.net/1911/15902.
URI: http://hdl.handle.net/1911/15902
Date: 1985

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