Kinetic analysis of RNA cleavage by coronavirus Nsp15 endonuclease: Evidence for acid base catalysis and substrate dependent metal ion activation.

Understanding the functional properties of SARS-CoV-2 nonstructural proteins is essential for defining their roles in the viral life cycle, developing improved therapeutics and diagnostics, and countering future variants. Coronavirus nonstructural protein Nsp15 is a hexameric U-specific endonuclease whose functions, substrate specificity, mechanism, and dynamics have not been fully defined. Previous studies report SARS-CoV-2 Nsp15 requires Mn ions for optimal activity; however, the effects of divalent ions on Nsp15 reaction kinetics have not been investigated in detail. Here, we analyzed the single and multiple turnover kinetics for model single-stranded RNA substrates. Our data confirm that divalent ions are dispensable for catalysis and show that Mn activates Nsp15 cleavage of two different ssRNA oligonucleotide substrates, but not a dinucleotide. Furthermore, biphasic kinetics of ssRNA substrates demonstrates that Mn stabilizes alternative enzyme states that have faster substrate cleavage on the enzyme. However, we did not detect Mn-induced conformational changes using CD and fluorescence spectroscopy. The pH-rate profiles in the presence and absence of Mn are consistent with active site ionizable groups with similar pKs of ca. 4.8-5.2. We found the Rp stereoisomer phosphorothioate modification at the scissile phosphate had minimal effect on catalysis, which supports a mechanism involving an anionic transition state. In contrast, the Sp stereoisomer is inactive due to weak binding, consistent with models that position the non-bridging phosphoryl oxygen deep in the active site. Together, these kinetic data demonstrate that Nsp15 employs a conventional acid-base catalytic mechanism passing through an anionic transition state, and that divalent ion activation is substrate-dependent.