Calcium inhibition of ribonuclease H1 two-metal ion catalysis

Abstract

Most phosphate-processing enzymes require Mg(2+) as a cofactor to catalyze nucleotide cleavage and transfer reactions. Ca(2+) ions inhibit many of these enzymatic activities, despite Ca(2+) and Mg(2+) having comparable binding affinities and overall biological abundances. Here we study the molecular details of the calcium inhibition mechanism for phosphodiester cleavage, an essential reaction in the metabolism of nucleic acids and nucleotides, by comparing Ca(2+)- and Mg(2+) catalyzed reactions. We study the functional roles of the specific metal ion sites A and B in enabling the catalytic cleavage of an RNA/DNA hybrid substrate by B. halodurans ribonuclease (RNase) H1 using hybrid quantum-mechanics/molecular mechanics (QM/MM) free energy calculations. We find that Ca(2+) substitution of either of the two active-site Mg(2+) ions substantially increases the height of the reaction barrier and thereby abolishes the catalytic activity. Remarkably, Ca(2+) at the A site is inactive also in Mg(2+)-optimized active-site structures along the reaction path, whereas Mg(2+) substitution recovers activity in Ca(2+)-optimized structures. Geometric changes resulting from Ca(2+) substitution at metal ion site A may thus be a secondary factor in the loss of catalytic activity. By contrast, at metal ion site B geometry plays a more important role, with only a partial recovery of activity after Mg(2+) substitution in Ca(2+)-optimized structures. Ca(2+)-substitution also leads to a change in mechanism, with deprotonation of the water nucleophile requiring a closer approach to the scissile phosphate, which in turn increases the barrier. As a result, Ca(2+) is less efficient in activating the water. As a likely cause for the different reactivities of Mg(2+) and Ca(2+) ions in site A, we identify differences in charge transfer to the ions and the associated decrease in the pKa of the oxygen nucleophile attacking the phosphate group.

Figure 1

(A) Details of the quantum region in the RNase H1 active site of the MgMg system (protein and nucleic acid: cartoon representation; classical water molecules: lines; quantum region: atomic representation). The total electron density difference between the CaMg and MgMg systems is drawn at the 0.01 contour level (blue; au). Whereas the large blue sphere at the metal A site reflects the larger number of electrons on calcium, the small blue regions on the coordinated oxygen atoms illustrate the smaller extent of charge transfer from these ligands to Ca²⁺ as compared to Mg²⁺ (see also Figure S6 in Supporting Information [SI]). (B) Active site of RNase H1 in complex with two Ca²⁺ ions. Alignment between the minimized reactant-state structure (B. halodurans, orange carbon backbone and Mg²⁺ ions) and the crystallographic structure (human, PDB ID: 2QKK, blue carbon backbone and green Mg²⁺ ions) is shown for side chains and substrate within 4 Å of the Mg²⁺. Water is omitted. (C) Schematics of the phosphodiester cleavage reaction catalyzed by RNase H1. In our calculations, we concentrate on the nucleophilic attack and phosphate–diester bond breaking (Step 1). Protonation of the ribose in Step 2 is not detailed here.

Figure 2

Reaction free energy and minimum energy profiles for single Ca²⁺ substitutions in RNase H1: (A) 2D free energy surface of the CaMg system projected on Q_e–Q_p plane (scale bar in kcal/mol). Q_e is defined as the difference between the bond-breaking and bond-forming P–O distances, Q_p is the proton transfer coordinate defined as the difference between the bond-breaking and bond-forming O–H distances (see also Figure S2 in SI). The converged string is shown in black. (B) 2D free energy surface of the MgCa system. The converged string is shown in black. Insets in A and B indicate the metal coordination geometry. (C) Energy minimized pathway for the CaMg system (red, triangles, labeled “Opt”). Q_ep is defined as the sum of Q_e and Q_p (Figure S2 in SI). Single-point QM/MM energies at the same level of theory are also shown for MgMg (blue, crosses, labeled “SP”) obtained by substituting Mg²⁺ for Ca²⁺ in energy minimized CaMg structures along the reaction pathway without further relaxation. (D) Energy minimized pathway for the MgCa system (green circles, labeled “Opt”). In (C) and (D), single-point QM/MM energies at the same level of theory are also shown for MgMg (blue, crosses, labeled “SP”) obtained by replacing Ca²⁺ with Mg²⁺ using the coordinates obtained from the CaMg energy minimized pathway, without further relaxation. Insets in C and D show TS structures, as indicated by the arrows.

Figure 3

Reaction pathways projected onto the distances of the oxygen atom of the nucleophilic water to the scissile phosphate (P–O, x-axis) and to the proton abstracted from the nucleophile (O–H, y-axis). The average distances of the last iteration in the string free energy simulations are shown for MgMg (blue) and CaMg (red). The initial pathways, before the string iterations, are shown as dashed lines. Product state (PS), transition state (TS), and reactant state (RS) distances are indicated.