pH Dependence of Catalysis by Pseudomonas aeruginosa Isochorismate–Pyruvate Lyase: Implications for Transition State Stabilization and the Role of Lysine 42

Abstract

An isochorismate-pyruvate lyase with adventitious chorismate mutase activity from Pseudomonas aerugionsa (PchB) achieves catalysis of both pericyclic reactions in part by the stabilization of reactive conformations and in part by electrostatic transition-state stabilization. When the active site loop Lys42 is mutated to histidine, the enzyme develops a pH dependence corresponding to a loss of catalytic power upon deprotonation of the histidine. Structural data indicate that the change is not due to changes in active site architecture, but due to the difference in charge at this key site. With loss of the positive charge on the K42H sidechain at high pH, the enzyme retains lyase activity at approximately 100-fold lowered catalytic efficiency, but loses detectable mutase activity. We propose that both substrate organization and electrostatic transition state stabilization contribute to catalysis. However, the dominant reaction path for catalysis is dependent on reaction conditions, which influence the electrostatic properties of the enzyme active site amino acid sidechains.

Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities

“…Since pericyclic reactions have a strong entropic/steric component to their energy barrier, it follows that enzymes that perform one reaction may have adventitious activity for the other. This has been documented previously for PchB (a lyase with adventitious mutase activity) (23, 25, 27, 46–48) , and here for Irp9 (an enzyme with physiological lyase activity and adventitious mutase activity). The adventitious pericyclic reactions were more readily measured and sometimes augmented in the variants tested.…”

Lysine221 is the general base residue of the isochorismate synthase from Pseudomonas aeruginosa (PchA) in a reaction that is diffusion limited

“…The coupled assay described above was again used. PchB has been shown previously to have constant activity throughout the pH range tested (25) . The secondary structure of PchA was monitored at the pH extremes and found to be intact by circular dichroism, but the protein was not stable at pH values below 6 or above 9 (data not shown).…”

Pericyclic Reactions Catalyzed by Chorismate-Utilizing Enzymes

“…Three structures of wildtype PchB have been published (9, 10) and these indicate that PchB is indeed a structural homologue of EcCM (Figure 2). In the structure of EcCM, an oxabicyclic transition state analogue is held in place by interaction with a total of eight charged/polar amino acids (11).…”

“…Importantly, mutation of the lysine at the 42 position (green in Figure 3) that is comparable to the lysine hypothesized to be important in electrostatic transition state stabilization in EcCM did not lead to structural perturbation of the active site. Evidence for this is provided in the form of circular dichroism data (7), and also the x-ray structures of the K42A (7) and K42E (9) mutants. However, K42A-PchB showed 1% of WT mutase and lyase activities, whereas the K42E mutant had no detectible activity for either reaction (7).…”

“…It should be noted that this variant form retains the ability to catalyze the lyase reaction when the sidechain is deprotonated (~100-fold decrease in k cat / K M ), indicating that the enzyme is a sufficient catalyst even without this sidechain charge (9). A calculation of an experimental ΔΔG ‡ by comparing the mutational effect at low and high pH gives a value of 2.4 kcal/mol for electrostatic transition state stabilization at the 42 site.…”