Insensitivity of Perturbed Carboxyl pKa Values in the Ovomucoid Third Domain to Charge Replacement at a Neighboring Residue

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Abstract
A number of carboxyl groups in turkey ovomucoid third domain (OMTKY3) have low pKa values. A previous study suggested that neighboring amino groups were primarily responsible for the low carboxyl pKa values. However, the expected elevation in pKa values for these amino groups was not observed. In the present study, site-directed mutagenesis is used to investigate the origins of perturbed carboxyl pKa values in OMTKY3. Electrostatic calculations suggest that Lys 34 has large effects, 0.4−0.6 unit, on Asp 7, Glu 10, and Glu 19 which are 5−11 Å away from Lys 34. Two-dimensional 1H NMR techniques were used to determine pKa values of the acidic residues in OMTKY3 mutants in which Lys 34 has been replaced with threonine and glutamine. Surprisingly, the pKa values in the mutants are very close to those of the wild-type protein. The insensitivity of the acidic residues to replacement of Lys 34 suggests that long-range electrostatic interactions play less of a role in perturbing carboxyl pKa values than originally thought. We hypothesize that hydrogen bonds play a key role in perturbing some of the carboxyl ionization equilibria in OMTKY3.

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