Differential Role of the Protein Matrix on the Binding of a Catalytic Aspartate to Mg2+ vs Ca2+: Application to Ribonuclease H

Divalent metal cations are essential cofactors for many enzyme functions. Although Mg2+ is the native cofactor in many enzymes such as ribonuclease H, its competitor Ca2+ may also bind to the enzyme but inhibit catalysis. Thus, the competition between Mg2+ and Ca2+ for a given metal-binding site in an enzyme and their effects on enzyme activity are of great interest. Most studies have focused on the interactions between Mg2+ or Ca2+ and the metal ligands in the first and sometimes second coordination shell. However, no study (to our knowledge) has examined the role of the protein architecture and surrounding aqueous environment on the binding of Mg2+ vs Ca2+ to a given protein metal-binding site. In this work, the free energy barriers for the binding of a catalytically essential aspartate to Mg2+ or Ca2+ in ribonuclease H from two organisms were computed using umbrella sampling with a classical force field (“classical” model). The corresponding free energy barriers in water were computed using the “classical” model as well as density functional theory combined with a self-consistent reaction field. The results reveal that, relative to water, the protein architecture and coupled protein–water interactions raise the free energy barrier for binding of the catalytically essential aspartate to the native Mg2+ cofactor more than the respective binding to Ca2+. They also reveal the physical basis for the different observed binding modes of Mg2+ and Ca2+ and highlight limitations of simulations with classical force fields that do not explicitly account for charge transfer and polarization effects.