Integration of kinetic isotope effect analyses to elucidate ribonuclease mechanism

Abstract

The well-studied mechanism of ribonuclease A is believed to involve concerted general acid–base catalysis by two histidine residues, His12 and His119. The basic features of this mechanism are often cited to explain rate enhancement by both protein and RNA enzymes that catalyze RNA 2’-O-transphosphorylation. Recent kinetic isotope effect analyses and computational studies are providing a more chemically detailed description of the mechanism of RNase A and the rate limiting transition state. Overall, the results support an asynchronous mechanism for both solution and ribonuclease catalyzed reactions in which breakdown of a transient dianoinic phosphorane intermediate by 5’O-P bond cleavage is rate limiting. Relative to non-enzymatic reactions catalyzed by specific base, a smaller KIE on the 5’O leaving group and a less negative βLG are observed for RNase A catalysis. Quantum mechanical calculations consistent with these data support a model in which electrostatic and H-bonding interactions with the non-bridging oxygens and proton transfer from His119 render departure of the 5'O less advanced and stabilize charge buildup in the transition state. Both experiment and computation indicate advanced 2’O-P bond formation in the rate limiting transition state. However, this feature makes it difficult to resolve the chemical steps involved in 2’O activation. Thus, modeling the transition state for RNase A catalysis underscores those elements of its chemical mechanism that are well resolved, as well as highlighting those where ambiguity remains.

Dynamical ensemble of the active state and transition state mimic for the RNA-cleaving 8–17 DNAzyme in solution

“…In the GB − and AP simulations, a bridging Na + ion plays an organizational role in bringing together the nucleophile and pro- R P NPO and facilitating in-line fitness (α catalysis), while also positioning the nucleophile (tertiary γ catalysis) so as to form a productive hydrogen bond interaction with the G13:N1 heteroatom required for nucleophile activation. In this position, the Na + ion also increases the acidity of the nucleophile to facilitate proton transfer to the general base (secondary γ catalysis), in a fashion similar to the role proposed for Lys41 in the classic RNA-cleavage mechanism of RNase A (55,75). Na + ions also engage in a second form of secondary γ catalysis by p K a tuning of G13:N1 through binding to the solvent-exposed Hoogsteen edge of the general base (G13:O6), similar to the recently proposed role of a Mg 2+ ion in the hammerhead ribozyme (76–78).…”

Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2

“…7 ). In RNase A the transphosphorylation reaction proceeds via an asynchronous concerted general acid/base mechanism involving His12, His119, and Lys41 23 . In this mechanism the 2′OH proton is transferred to the deprotonated form of His12 to activate the 2′O nucleophile.…”

Beyond the Plateau: pL Dependence of Proton Inventories as a Tool for Studying Ribozyme and Ribonuclease Catalysis

“…Acid/base catalysis is a ubiquitous strategy employed by ribonucleases − and small ribozymes − to stabilize the transition states for RNA strand cleavage by 2′-O-transphosphorylation (Figure ). , In a few well-studied examples, information from high-resolution structures and biophysical, biochemical, and kinetic data have come together to identify active site functional groups involved in proton transfer for both RNA and protein enzymes. − However, in most cases, a complete description of the number and precise role of active site functional groups involved in acid/base catalysis is lacking.…”