Thermodynamic linkage between the binding of protons and inhibitors to HIV-1 protease

The aspartyl dyad of free HIV-1 protease has apparent pK(a)s of similar to 3 and similar to 6, but recent NMR studies indicate that the aspartyl dyad is fixed in the doubly protonated form over a wide pH range when cyclic ure a inhibitors are bound, and in the monoprotonated form when the inhibitor K NI-272 is bound. We present computations and measurements related to these changes in protonation and to the thermodynamic linkage between protonation and inhibition. The Poisson-Boltzmann model of electrostatics is used to c ompute the apparent pK(a)s of the aspartyl dyad in the free enzyme and in c omplexes with four different inhibitors. The calculations are done with two parameter sets. One assigns epsilon = 4 to the solute interior and uses a detailed model of ionization; the other uses epsilon = 20 for the solute in terior and a simplified representation of ionization. For the free enzyme, both parameter sets agree well with previously measured apparent pK(alpha)s of similar to 3 and similar to 6. However, the calculations with an intern al dielectric constant of 4 reproduce the large pK(a) shifts upon binding o f inhibitors, but the calculations with an internal dielectric constant of 20 do not. This observation has implications for the accurate calculation o f pK(a)s in complex protein environments. Because binding of a cyclic urea inhibitor shifts the pK(a)s of the asparty l dyad, changing the pH is expected to change its apparent binding affinity . However, we find experimentally that the affinity is independent of pH fr om 5.5 to 7.0. Possible explanations for this discrepancy are discussed.