QM/MM Simulation (B3LYP) of the RNase A Cleavage-Transesterification Reaction Supports a Triester AN+DN Associative Mechanism with an O2´ H Internal Proton Transfer

Journal Article · Wed Jan 22 04:00:00 EST 2014 · Journal of the American Chemical Society

DOI:https://doi.org/10.1021/ja406122c· OSTI ID:1233781

Elsasser, Brigitta M.; Fels, Gregor; Weare, John H.

The mechanism of the backbone cleavage transesterification step of the RNase A enzyme remains controversial even after 60 years of study. We report QM/MM free energy calculations for two optimized reaction paths based on an analysis of all structural data and identified by a search for reaction coordinates using a reliable quantum chemistry method (B3LYP), equilibrated structural optimizations, and free energy estimations. Both paths are initiated by nucleophilic attack of the ribose O2? oxygen on the neighboring diester phosphate bond and both reach the same product state (PS) (a O3??O2? cyclic phosphate and a O5? hydroxyl terminated fragment). Path 1, resembles the widely accepted dianionic transition state (TS) general acid (His 119)/base (His 12) classical mechanism. However, this path has a barrier (25 kcal/mol) higher than that of the rate limiting hydrolysis step and a very loose TS. In Path 2, the proton initially coordinating the O2? migrates to the non-bridging O1P in the initial reaction path rather than directly to the general base resulting in a triester (substrate as base) AN+DN mechanism with a monoanionic weakly stable intermediate. The structures in the transition region are associative with low barriers (TS1 10, TS2 7.5 kcal/mol). The Path 2 mechanism is consistent with the many results from enzyme and buffer catalyzed and uncatalyzed analog reactions and leads to a PS consistent with the reactive state for the following hydrolysis step. The differences between the consistently estimated barriers in Path 1 and 2 lead to a 1011 difference in rate strongly supporting the less accepted triester mechanism.

• Research Organization: Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 1233781
• Journal Information: Journal of the American Chemical Society, Journal Name: Journal of the American Chemical Society Journal Issue: 3 Vol. 136; ISSN 0002-7863
• Publisher: American Chemical Society (ACS)
• Country of Publication: United States
• Language: English

Related Subjects

Environmental Molecular Sciences Laboratory