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Unclenching Chorismate Lynse: Effects of the C90A Active Site Mutation and of Binding the Inhibitor Vanillate

Published

Author(s)

David Travis Gallagher, N Smith, M P. Mayhew, Marcia J. Holden

Abstract

Chorismate Lyase from E. coli is strongly product inhibited retaining the product 4-hydroxybenzoate internally by extensive hydrogen bonding.The biological rationale for this unusual retention, and the mechanism of release, may involve pathway regulation and/or the controlled transfer of the product to the membrane-bound receiving enzyme. Release of the product appears to require the opening of two heliz-turn-helix flaps that overlie the active site. We have tested several modifications to the enzyme in order to study the catalytic mechanism and product release process. Replacing the active site Gly 90 with alanine gives slightly reduced kinetic turnover and tighter product binding. We report the crystal structures of the G90A mutant in complex with product and with the inhibitor vanillate, whose size is intermediate between the product and the larger substrate. The structure shows two orientations of the vanillate molecule in the active site, several new buried water sites, discrete disorder of proximal sidechains, and a second internal site at which vanillate appears to bind. Also reported is the structure of the wild-type active site with bound vanillate, in which the active site flaps are partly opened. Comparison of these structures with the wild-type active site product complex enables structural and dynamic inferences regarding flap motions and by extension, the product release mechanism.
Citation
Biochemistry

Keywords

crystal structure, discrete disorder, parahydroxy benzoate, product inhibition, protein engineering

Citation

Gallagher, D. , Smith, N. , Mayhew, M. and Holden, M. (2008), Unclenching Chorismate Lynse: Effects of the C90A Active Site Mutation and of Binding the Inhibitor Vanillate, Biochemistry (Accessed July 21, 2024)

Issues

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Created October 16, 2008