Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.352628
Title: Aspects of the structure and catalytic function of the arom multifunctional enzyme from Neurospora crassa
Author: Boocock, Martin R.
ISNI:       0000 0001 3470 2148
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1983
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Abstract:
1. The established methods for the isolation of the arom multifunctional enzyme from N.crassa have been modified to take into account the instability of two of the component activities. The copurification in constant ratio of the five activities of the enzyme complex has been demonstrated. 2. The homogeneous multifunctional enzyme isolated by the modified procedures contains approximately 1 atom/subunit of tightly bound zinc, which is essential for DHQ synthase (El) activity. DHQ synthase activity is very rapidly reconstituted on addition of the inactive EDTA-treated enzyme to zinc ion "buffering" systems. 3. Chorismate synthase and anthranilate synthase suitable for use as coupling enzymes in steady state kinetics experiments have been isolated from N.crassa. Chorismate synthase has been purified to electrophoretic homogeneity. The catalytic properties of the purified enzyme are qualitatively different from those of the highly purified chorismate synthase preparation of Welch et al (1974). 4. The catalytic properties of chorismate synthase isolated from N.crassa strongly suggest that the enzyme is bifunctional, and that reduced FMN participates in the conversion of EPSP to chorismate. 5. A detailed study of the steady state kinetic properties of the EPSP synthase (E5) activity of the arom enzyme complex has been undertaken. The results support an ordered sequential mechanism in which shikimate 3-phosphate is the first substrate to bind to the enzyme in the forward reaction; EPSP and Pi bind to the enzyme in a random order in the reverse reaction. 6. Arsenate can substitute for phosphate in the reverse reaction of EPSP synthase. Kinetic experiments using arsenate as a pseudo-substrate indicate that the release of enzyme-bound EPSP is the rate-limiting step in the forward reaction. 1. The herbicide glyphosate (N-phosphonomethyl glycine) is a potent reversible inhibitor of the EPSP synthase activity of the arom enzyme complex. Glyphosate interacts uniquely with an enzyme o shikimate 3-phosphate kinetic complex, and excludes productive binding of PEP. The steady state kinetic properties of the arom EPSP synthase strongly suggest that glyphosate mimics the enzyme-bound conformation of the natural substrate, PEP. 8. The equilibrium constant of the EPSP synthase reaction, re-estimated at pH 7.0 under the conditions used in the steady state kinetics experiments, is 116. 9. The structural organisation of the arom multifunctional enzyme has been investigated by limited proteolysis, and catalytically active fragments have been resolved by chromatography under nondenaturing conditions. In particular, the products of limited trypsin/chymotrypsin digestion of the enzyme complex have been separated into active fragments carrying E5 activity and E2/E3 activities. These fragments have been isolated and characterised, and are derived from non-overlapping sub-regions of the arom polypeptide. 10. The limited proteolysis results are best interpreted in terms of a "gene fusion" model for the origin of the arom multifunctional enzyme.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.352628  DOI: Not available
Keywords: Fungal enzyme analysis
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