Chorismate Lyase: Kinetics and Engineering for Stability

Published

January 31, 2002

Author(s)

Marcia J. Holden, M P. Mayhew, David T. Gallagher, V L. Vilker

Abstract

By removing the enolpyruvyl group from chorismate, chorismate lyase (CL) produces p-hydroxybenzoate (p-HB) for the ubiquinone biosynthetic pathway. We have analyzed CL by several spectroscopic and chemical techniques and measured its kinetic (k_cat = 1.7x^-1, K_m = 29 M) and product inhibition parameters (K_p = 2.1 M for p-HB). Protein aggregation, a serious problem with wild type CL, proved to be primarly due to the presence of two surface-active cysteines, whose chemical modification or mutation (to serines) gave greatly improved solution behavior and minor effects on enzyme activity. CL is strongly inhibited by its product p-HB; for this reason activity and inhibition measurements were analyzed by both initial rate and progress curve methods. The results are consistent, but in this case where the stable enzyme-product complex rapidly becomes the predominant form of the enzyme, progress curve methods are more efficient. We also report inhibition measurements with several substrate and product analogs that give information on ligand binding interactions of the active site. The biological function of the unusual product retention remains uncertain, but may involve a mechanism of directed delivery to the membrane-bound enzyme that follows CL in the ubiquinone pathway.

Citation

Biochimica Et Biophysica ACTA-Protein Structure and Molecular Enzymology

Volume

1594

Issue

1

Pub Type

Journals

Keywords

5, 5'-dithiobis 2-nitrobenzoic acid (DTNB, chorismate, circular dichrosim, p-hydroxybenzoate, product inhibition, progress curve analysis

Citation

Holden, M. , Mayhew, M. , Gallagher, D. and Vilker, V. (2002), Chorismate Lyase: Kinetics and Engineering for Stability, Biochimica Et Biophysica ACTA-Protein Structure and Molecular Enzymology (Accessed April 19, 2024)