Chorismate lyase: kinetics and engineering for stability.
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 (kcat=1.7 s(-1), K(m)=29 microM) and product inhibition parameters (K(p)=2.1 microM for p-HB). Protein aggregation, a serious problem with wild type CL, proved to be primarily 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.
Holden
M J
MJ
National Institute of Standards and Technology, Chemical Science and Technology Laboratory, Biotechnology Division, 100 Bureau Drive, Gaithersburg, MD 20899-8312, USA. marcia.holden@nist.gov
Mayhew
M P
MP
Gallagher
D T
DT
Vilker
V L
VL
D000878:Anthranilate Synthase / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002826:Chorismate Mutase / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D007652:Oxo-Acid-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D010226:Parabens / Q000737:chemistry; D015202:Protein Engineering; D012694:Serine / Q000737:chemistry; D012995:Solubility; D014451:Ubiquinone / Q000737:chemistry
D010226:Parabens; D014451:Ubiquinone; D012694:Serine; D007652:Oxo-Acid-Lyases; C075960:chorismate pyruvate lyase; D000878:Anthranilate Synthase; D002826:Chorismate Mutase; C038193:4-hydroxybenzoic acid; D003545:Cysteine