Use of 1H NMR spectroscopy and computer simulations To analyze histidine pKa changes in a protein tyrosine phosphatase: experimental and theoretical determination of electrostatic properties in a small protein.

In bovine low Mr protein tyrosine phosphatase, the pKa values of His-66 and His-72 are 8.3 and 9.2, respectively. These unusually high values were hypothesized to be caused by electrostatic interactions with several nearby negatively charged groups. To test this, mutant enzymes were made in which one or more carboxylate side chains were removed or introduced near the histidines. Michaelis kinetic parameters, measured using p-nitrophenyl phosphate as a substrate, indicated that all mutant enzymes retained approximately 50% or more of the activity of wild-type enzyme. The effect that each mutation had on the pKa of the nearby histidine was monitored by 1H NMR spectroscopy using the MLEV-17 pulse sequence to filter out the broad interfering amide resonances in the spectra. Independently, computer simulations of the pKas were obtained using the finite difference method to solve the linear Poisson-Boltzmann equation. The proximity of a charged residue to the titrating histidine imidazole largely determined the extent of the pKa perturbation. The change in pKa for His-72 in the mutant enzymes was -1.69 units for D42A, -2.36 units for E23A, -2.99 units for E23A/D42A, and unchanged for E139A and Q143E. Thus, the pKa of His-72 in the double mutant E23A/D42A decreased to nearly that of a free histidine imidazole group. The His-66 pKa change was -1.25 units for E139A and was not significant for the other mutants. His-66, Glu-139, and Gln-143 are at the protein surface and much more exposed to the higher solvent dielectric compared to His-72, Glu-23, and Asp-42. These structural characteristics explain the smaller decrease in the observed pKa of His-66 for the E139A mutant compared to the decrease in the pKa of His-72 when a single nearby carboxylate was removed. These observations were adequately predicted by theoretical electrostatic calculations using the Poisson-Boltzmann equation as a model for a solute molecule of low dielectric in solution of high dielectric.