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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.

SUBMITTER: Rosta E

PROVIDER: S-EPMC3985467 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Calcium inhibition of ribonuclease H1 two-metal ion catalysis.

Rosta Edina E Yang Wei W Hummer Gerhard G

Journal of the American Chemical Society 20140218 8

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. ...[more]

PMID: 24499076

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Project description:Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase superfamily and hydrolyzes the phosphodiester linkage on the RNA strand of a DNA/RNA hybrid duplex. Due to its activity in HIV reverse transcription, it represents a promising target for anti-HIV drug design. While crystallographic data have located two ions in the catalytic site, there is ongoing debate concerning just how many metal ions bound at the active site are optimal for catalysis. Indeed, experiments have shown a dependency of the catalytic activity on the Mg(2+) concentration. Moreover, in RNase H, the glutamate residue E188 has been shown to be essential for full enzymatic activation, regardless of the Mg(2+) concentration. The catalytic center is known to contain two Mg(2+) ions, and E188 is not one of the primary metal ligands. Herein, classical molecular dynamics (MD) simulations are employed to study the metal-ligand coordination in RNase H at different concentration of Mg(2+). Importantly, the presence of a third Mg(2+) ion, bound to the second-shell ligand E188, is a persistent feature of the MD simulations. Free energy calculations have identified two distinct conformations, depending on the concentration of Mg(2+). At standard concentration, a third Mg(2+) is found in the catalytic pocket, but it does not perturb the optimal RNase H active conformation. However, at higher concentration, the third Mg(2+) ion heavily perturbs the nucleophilic water and thereby influences the catalytic efficiency of RNase H. In addition, the E188A mutant shows no ability to engage additional Mg(2+) ions near the catalytic pocket. This finding likely explains the decrease in catalytic activity of E188A and also supports the key role of E188 in localizing the third Mg(2+) ion at the active site. Glutamate residues are commonly found surrounding the metal center in the endonuclease family, which suggests that this structural motif may be an important feature to enhance catalytic activity. The present MD calculations support the hypothesis that RNase H can accommodate three divalent metal ions in its catalytic pocket and provide an in-depth understanding of their dynamic role for catalysis.

Dataset Information

Calcium inhibition of ribonuclease H1 two-metal ion catalysis.

Publications

Calcium inhibition of ribonuclease H1 two-metal ion catalysis.

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