Nucleophile activation in PD…(D/E)xK metallonucleases: An experimental and computational pKa study

Metallonucleases conduct metal-dependent nucleic acid hydrolysis. While metal ions serve in multiple mechanistic capacities in these enzymes, precisely how the attacking water is activated remains unclear for those lacking an obvious general base. All arguments hinge on appropriate pKas for active site moieties very close to this species, and measurement of the pKa of a specific water molecule is difficult to access experimentally. Here we describe a computational approach for exploring the local electrostatic influences on the water-derived nucleophile in metallonucleases featuring the common PD…(D/E)xK motif. We utilized UHBD to predict the pKas of active site groups, including that of a water molecule positioned to act as a nucleophile. The pKa of a Mg(II)-ligated water molecule hydrogen bonded to the conserved Lys70 in a Mg(II)–PvuII enzyme complex was calculated to be 6.5. The metal and the charge on the Lys group were removed in separate experiments; both resulted in the elevation of the pKa of this water molecule, consistent with contributions from both moieties to lowering this pKa. This behavior is preserved among other PD…(D/E)xK metallonucleases. pKas extracted from the pH dependence of the single turnover rate constant are compared to previous experimental data and the above predicted pKas.

Graphical abstractUniv. of Houston Brownian Dynamics was used to explore the electrostatic influences on the water-derived nucleophile in metallonucleases featuring the PD…(D/E)xK motif. Removal of the conserved Lys charge resulted in pKa elevation of this water molecule in a number of these enzymes, consistent with a contribution to lowering this pKa.Figure optionsDownload full-size imageDownload as PowerPoint slide