Studies of the Structure and Catalytic Mechanism of Chorismate Synthase

White, Peter J (1988) Studies of the Structure and Catalytic Mechanism of Chorismate Synthase. PhD thesis, University of Glasgow.

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1. The established methods for the purification of the N. crassa chorismate synthase have been modified to overcome proteolytic damage and to obtain electrophoretically homogeneous enzyme free from substrates. 2. The purified N. crassa chorsmate synthase is a homotetramer with a subunit Mr of 50 000. The enzyme is bifunctional, showing both chorismate synthase and flavin reductase activity. 3. A simple anaerobic assay procedure has been developed which enables chorismate synthase activity to be monitored under strict anaerobic conditions. This procedure was used to detect and purify chorismate synthase from an overproducing strain of E. coli. 4. The purified E. coli chorismate synthase is a homotetramer with a subunit Mr of 38 000. The enzyme is active only in the presence of exogeneously supplied reduced flavin and appears to lack the ability to reduce this cofactor intrinsically. 5. The N-terminal sequence of the E. coli chorismate synthase has been determined for the first thirty residues and matches exactly that predicted from the nucleotide sequence. The complete amino acid sequence of the aroC structural gene is confirmed. The E. coli chorismate synthase snows primary structure homology with internal sequences derived from peptides isolated from the N. crassa enzyme. 6. Gross overexpression of the E. coli cnonsmate synthase using an IPTG induced overproducing strain resulted in the concomitant overproduction of a yellow chromophore. Spectroscopic and enzymatic analyses indicate that this chromophore is FMN. The E. coli enzyme contains limited amounts of residually bound flavin, as determined by fluorescence spectroscopy. Both the N. crassa and E. coIi chorismate synthase have a preference for FMN over FAD as the flavin cofactor. A putative FMN-phosphate binding region is identified from the E. coli chorismate synthase amino acid sequence. 7. Analysis of aromatic and flavin fluorescence spectra indicate that in vivo tne E. coli chorismate synthase subunit is the binding site for FMN. The N. crassa enzyme is nighly sensitive to thiol directed agents and insensitive to metal activation or metal chelation. 8. All of the structural and kinetic analyses presented suggest that the N. crassa enzyme is a fusion protein; a chorismate synthase domain coupled to a flavin reductase activity. 9. Preliminary evidence suggests that neither a thiol nor a metal is involved at the chorismate synthase active site, but that flavin itself could adequately assume the role of a nucleophile in a two-step reaction mechanism.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biochemistry
Date of Award: 1988
Depositing User: Enlighten Team
Unique ID: glathesis:1988-77710
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 11:53
Last Modified: 14 Jan 2020 11:53

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