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Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

Title
Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction
Authors
Lopata, AnnaJambrina, Pablo G.Sharma, Pankaz K.Brooks, Bernard R.Toth, JuditVertessy, Beata G.Rosta, Edina
Ewha Authors
Pankaz Kumar Sharma
Issue Date
2015
Journal Title
ACS CATALYSIS
ISSN
2155-5435JCR Link
Citation
ACS CATALYSIS vol. 5, no. 6, pp. 3225 - 3237
Keywords
QM/MM (quantum mechanics/molecular mechanics)dUTPasecontinuous symmetry measureone-metal ion catalytic mechanismab initioDFT (density functional theory)coupled proton transfer
Publisher
AMER CHEMICAL SOC
Indexed
SCIE; SCOPUS WOS scopus
Document Type
Article
Abstract
Most enzymes present a catalytic mechanism where one or more proton transfer events occur coupled tightly together with the enzymatic chemical reaction. We show here that inactivating mutations decouple this proton transfer step from the phosphate cleavage reaction in dUTPase. Homotrimeric dUTPase enzymes catalyze the hydrolysis of dUTP to dUMP and pyrophosphate, using largely similar structural and functional groups as most AAA+ enzymes. dUTPases typically use a single Mg2+ ion as a cofactor in the active site that is formed by direct protein protein contacts including all three protomers. Here we focus on the C-terminal arm structural motif, which has sequence and functional similarities to P-loop motifs and is required for catalysis. In this work, we have studied the functional roles of the C-terminal arm in ligand binding and catalysis by using QM/MM (quantum mechanics/molecular mechanics) calculations in conjunction with site-directed mutagenesis experiments. We also present a new method to assess the metal ion coordination symmetry during the catalytic reaction. Using this new implementation, we identified that the coordination symmetry follows a consistent pattern in the three systems studied, reaching the most symmetrical state near the transition states. We found that the phosphate cleavage proceeds with a concerted bimolecular (A(N)D(N)) mechanism with a loose dissociative transition state and that it is coupled with a proton transfer step involving a unanimously conserved Asp residue. We show that the main mechanistic effect of the lack of the C-terminal ann is to decouple the phosphate cleavage from the subsequent proton transfer step, resulting in a high-barrier altered reaction pathway.
DOI
10.1021/cs502087f
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연구기관 > 약학연구소 > Journal papers
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