Functional tuning of the catalytic residue pK in a de novo designed esterase.

AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p-nitrophenyl acetate (pNPA) and 4-nitrophenyl-(2-phenyl)-propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single-histidine residue (His ) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pK value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pK calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pK value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pK value of this residue in water. We determined that a short-range favorable interaction with Glu contributes to the elevated pK of His . We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pK of its active nucleophile, His , by 0.7 units. As a direct result of the decrease in the His pK value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine-promoted catalysis. Proteins 2017; 85:1656-1665. © 2017 Wiley Periodicals, Inc.