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.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/15902 |
| Date | January 1985 |
| Creators | GREENHUT, JOAN |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | application/pdf |
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