title: Modification of residue 42 of the active site loop with a lysine-mimetic side chain rescues isochorismate-pyruvate lyase activity in Pseudomonas aeruginosa PchB.
abstract: PchB is an isochorismate-pyruvate lyase from Pseudomonas aeruginosa. A positively charged lysine residue is located in a flexible loop that behaves as a lid to the active site, and the lysine residue is required for efficient production of salicylate. A variant of PchB that lacks the lysine at residue 42 has a reduced catalytic free energy of activation of up to 4.4 kcal/mol. Construction of a lysine isosteric residue bearing a positive charge at the appropriate position leads to the recovery of 2.5-2.7 kcal/mol (about 60%) of the 4.4 kcal/mol by chemical rescue. Exogenous addition of ethylamine to the K42A variant leads to a neglible recovery of activity (0.180 kcal/mol, roughly 7% rescue), whereas addition of propylamine caused an additional modest loss in catalytic power (0.056 kcal/mol, or 2% loss). This is consistent with the view that (a) the lysine-42 residue is required in a specific conformation to stabilize the transition state and (b) the correct conformation is achieved for a lysine-mimetic side chain at site 42 in the course of loop closure, as expected for transition-state stabilization by the side chain ammonio function. That the positive charge is the main effector of transition state stabilization is shown by the construction of a lysine-isosteric residue capable of exerting steric effects and hydrogen bonding but not electrostatic effects, leading to a modest increase of catalytic power (0.267-0.505 kcal/mol of catalytic free energy, or roughly 6-11% rescue).
authors.affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA.
authors.forename: José
authors.initials: J
authors.lastname: Olucha
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Meneely
authors.forename: Audrey L
authors.initials: AL
authors.lastname: Lamb
mesh_terms: D001483:Base Sequence; D019757:Carbon-Oxygen Lyases / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D017931:DNA Primers; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008958:Models, Molecular; D018716:Molecular Mimicry; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013816:Thermodynamics
chemical_list: D017931:DNA Primers; D019757:Carbon-Oxygen Lyases; D008239:Lysine