Modification of residue 42 of the active site loop with a lysinemimetic sidechain rescues isochorismate-pyruvate lyase activity in Pseudomonas aeruginosa PchB

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Issue Date
2012-09-25Author
Olucha, José
Meneely, Kathleen M.
Lamb, Audrey L.
Publisher
ACS
Type
Article
Article Version
Scholarly/refereed, author accepted manuscript
Rights
Copyright © 2012 American Chemical Society
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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 sidechain 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).
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Citation
Olucha, J., Meneely, K. M., & Lamb, A. L. (2012). Modification of residue 42 of the active site loop with a lysine-mimetic sidechain rescues isochorismate-pyruvate lyase activity in Pseudomonas aeruginosa PchB. Biochemistry, 51(38), 7525–7532. http://doi.org/10.1021/bi300472n
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