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Multiple proton relay routes in the reaction mechanism of RNAP II: Assessing the effect of structural model
Author(s) -
Zhang Rui,
Bhattacharjee Anirban,
Field Martin J.,
Salahub Dennis R.
Publication year - 2015
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.24732
Subject(s) - pyrophosphate , chemistry , proton , active site , molecular dynamics , protein data bank (rcsb pdb) , yield (engineering) , reaction mechanism , substrate (aquarium) , density functional theory , catalysis , stereochemistry , enzyme , computational chemistry , physics , biochemistry , thermodynamics , oceanography , quantum mechanics , geology
ABSTRACT RNA polymerase II catalyzes the nucleotidyl transfer reaction for messenger RNA synthesis in eukaryotes. Two crystal structures of this system have been resolved, each with its own defects in the coordination sphere of Mg 2+ (A) resulting from chemical modifications. We have used both structures and also a novel hybrid of the two that allows a better exploration of the parts of configuration space that reflect substrate–enzyme interactions. MD and QM/MM calculations have been performed, the latter with the semiempirical AM1/d‐PhoT method, calibrated against density functional theory. Reaction path scans in 1‐D provided insights about the role of Mg 2+ (A) which turns out to be more structural than catalytic. In contrast, 1‐D scans of the incorporation of the nucleotidyl group yielded barriers that were much too high, necessitating the use of 2‐D reaction coordinates. Three different proton acceptors for the initial reaction step were examined. For those models based on the two PDB structures the 2‐D scans continued to yield very high barriers, indicating that the reaction is unlikely to proceed from these configurations. On the other hand, two hybrid models, chosen from the early and late parts of a 12ns molecular dynamics simulation yielded greatly reduced barriers in the range of ∼17 to ∼27 kcal/mol for the three proton acceptors, as compared to the experimental estimate of 18 kcal/mol. The final step, release of pyrophosphate, was found to be facile. Our overall mechanism involves only active site residues or water without the need for external reactive agents such as the hydroxide ion previously proposed. Proteins 2015; 83:268–281. © 2014 Wiley Periodicals, Inc.