Introducing a single mutation into the hydrophobic pocket of calmodulin confers catalytic activity to this non-enzymatic protein. A M144H mutation converts calmodulin into AlleyCatE, an efficient esterase, and a F92E mutation produces Kemp eliminase (AlleyCat). We employed NMR spectroscopy to measure pKa values of catalytic His144 and Glu92 residues in AlleyCatE and AlleyCat, respectively. We then rationally modulated local electrostatic potential in AlleyCatE to decrease the pKa of its active nucleophile, His144, by 0.7 units. Such change resulted in a 50% increase in catalytic efficiency at pH 6. This work shows that a series of simple NMR experiments that can be done using a low field spectrometer combined with straightforward computational analysis provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine-promoted catalysis. In the case of AlleyCat, a Kemp eliminase, the pKa value of the active Glu92 changes dramatically when substrate is present in the pocket. Without substrate, we measured pKa of 5.1, while in the presence of substrate-like inhibitor this value increased to 7.7. The pKa value obtained for E92 from NMR experiments is significantly depressed relative to the value (pKa = 6.9) previously reported based on pH-rate profile. These results show that the pKa values of the active site residues obtained from pH reactivity profiles must be interpreted with caution and direct NMR measurements provide a basis for rational improvement of catalytic properties of de novo designed catalysts.

Determinants of the pKa value of catalytic residue in de novo designed enzymes

253rd National Meeting (2017)

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