Altering the Y137-K164-K166 triad of mandelate racemase and its effect on the observed pKa of the Brønsted base catalysts

Abstract

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelate using a two-base mechanism with Lys 166 acting as the Brønsted base to abstract the α-proton from (S)-mandelate. The resulting intermediate is subsequently re-protonated by the conjugate acid of His 297 to yield (R)-mandelate. The roles of these amino acids are reversed when (R)-mandelate is the substrate. The side chains of Tyr 137, Lys 164, and Lys 166 form a H-bonding network and the proximity of the two ε-NH₃⁺ groups is believed to lower the pK_a of Lys 166. We used site-directed mutagenesis, kinetics, and pH-rate studies to explore the roles of Lys 164 (K164 C/M) and Tyr 137 (Y137 L/F/S/T) in catalysis. The efficiency (k_cat/K_m) was reduced ∼3.5 × 10⁵-fold for K164C MR, relative to wild-type MR, indicating a major role for this residue in catalysis. The efficiency of Y137F MR, however, was reduced only 25-30-fold. pH-Rate profiles (log k_cat vs. pH) revealed that substitution of Tyr 137 by Phe increased the kinetic pK_a of Lys 166 from 5.88 ± 0.02 to 7.3 ± 0.2. Hence, Tyr 137 plays an important role in facilitating the reduction of the pK_a of the Brønsted base Lys 166 by ∼1.4 units. Interestingly, the Phe substitution also increased the kinetic pK_a of His 297 from 5.97 ± 0.04 to 7.1 ± 0.1. Thus, the Tyr 137-Lys 164-Lys 166 H-bonding network plays a broader role in modulating the pK_a of catalytic residues by influencing the electrostatic character of the entire active site, not only by decreasing the observed pK_a value of Lys 166, but also by decreasing the pK_a of His 297 by 1.1 units.

Keywords: Enolase superfamily; Inhibition; Mandelate racemase; Site-directed mutagenesis; pH-rate profiles; pK(a) perturbation.