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 pKa 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 (kcat/Km) 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 kcat vs. pH) revealed that substitution of Tyr 137 by Phe increased the kinetic pKa 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 pKa of the Brønsted base Lys 166 by ∼1.4 units. Interestingly, the Phe substitution also increased the kinetic pKa 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 pKa of catalytic residues by influencing the electrostatic character of the entire active site, not only by decreasing the observed pKa value of Lys 166, but also by decreasing the pKa of His 297 by 1.1 units.