DETERMINATION OF PKAS OF IONIZABLE GROUPS IN PROTEINS - THE PKA OF GLU-7 AND GLU-35 IN HEN EGG-WHITE LYSOZYME AND GLU-106 IN HUMAN CARBONIC ANHYDRASE-II

We report a method by which molecular dynamics-free energy perturbation simulations can be used to estimate the pK(a) of ionizable groups in proteins. The method has been tested to demonstrate its effectiveness in determining the pK(a) of Glu 7 and 35 in hen egg white lysozyme, where the former has a pK(a) of 2.6 and the latter a perturbed pK(a) of 6.0-6.5 (in the presence of un-ionized Asp 52). We predict that the pK(a) of Glu 7 is 3.1 +/- 3.1, while we predict that Glu 35 has a pK(a) of 12.0 +/- 1.3. These test simulations indicate that (1) our approach is capable of predicting the perturbation of the pK(a) of glutamic acid to higher values, (2) the precision of the approach depends on the conformational flexibility of the glutamic acid side chain, and (3) the accuracy of the approach, on average, is +/- 3 pK(a) units. This approach was then applied to the problem of the pK(a) of Glu 106 in human carbonic anhydrase II (HCAII). The activity of HCAII is dependent on a group whose pK(a) is around 7.0. Glu 106 has been implicated as this group, but this requires the pK(a) of this residue to be around 7. We predict that this group has a pK(a) of 2.2 +/- 2.8, which, even given the accuracy of our method, suggests that this group is not the activity-linked group. The present work demonstrates that our approach can be fruitfully applied to chemically important questions and that free energy methods can be applied to the determination of pK(a)'s in proteins with an accuracy to about +/- 3 pK(a) units.