PH-DEPENDENCE OF BINDING REACTIONS FROM FREE-ENERGY SIMULATIONS AND MACROSCOPIC CONTINUUM ELECTROSTATIC CALCULATIONS - APPLICATION TO 2'GMP3'GMP BINDING TO RIBONUCLEASE T-1 AND IMPLICATIONS FOR CATALYSIS/

An approach is described for extending free energy calculations to tak e into account the pH dependence of the relative binding of ligands to an enzyme or other receptor protein. The method is based on the calcu lation of the free energy difference for a single protonation state vi a the thermodynamic cycle simulation approach followed by inclusion of all possible protonation states of the enzyme and the inhibitor by us e of a macroscopic continuum dielectric (Poisson-Boltzmann) model. A d etailed formulation of the combined model is presented. It involves so lution of the multiple equilibrium problem and makes use of the calcul ated pK(a) values of all titrating groups on both enzyme and ligand. T he method is illustrated by calculations of the pH dependence of the d ifferential binding of the inhibitors 2'GMP and 3'GMP to ribonuclease T-1. A free energy simulation of the differential binding is made for a given protonation state of the enzyme and inhibitor. Although only q ualitative agreement with experiment is obtained, the results provide insights concerning the interactions involved. The pH dependence of th e binding is calculated by using the protonation state of the residues from the free energy simulation as the standard state for a Poisson-B oltzmann calculation. Information is obtained concerning the pK(a) val ues of the titrating amino acids in the free, 2'GMP and 3'GMP bound en zyme forms of RNase T-1 and the difference in the pH dependence of the binding of 2'GMP and 3'GMP to RNase T-1. The contributions of differe nt types of interactions (e.g. protein residues versus solvent) to the free energy differences are examined. A free energy simulation of the pK(a) shift of Glu58 shows that it is important to consider both carb oxyl oxygen atoms as possible protonation sites since they may behave very differently in a protein. It is found in the protein that the int eractions with the solvent favor the neutral (protonated) state of Glu 58. This contrasts sharply with the solution behavior, where the solve nt favors the charged state. Analysis of the results shows that the in teractions of bound water with other protein residues leads to the obs erved effect. Comparisons are made with a continuum calculation that u ses the charged state employed in the free energy simulation. Implicat ions of the calculations and results for the catalytic mechanism of RN asc T-1 are outlined. An analysis is given of the stronger binding of 2'GMP,: which is more similar to the cyclic phosphate transition state , than 3'GMP. The pK(a) calculations support the role of His92 as the general acid in donating a proton to the 5' leaving group and the role of Glu58 as the general base involved in the extraction of a proton f rom the 2' hydroxyl of the substrate, a necessary step for the formati on of the pentacovalent transition state.