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Conformational change of the methionine 20 loop of Escherichia coli dihydrofolate reductase modulates pK a of the bound dihydrofolate
Author(s) -
Khavrutskii Ilja V.,
Price Daniel J.,
Lee Jinhyuk,
Brooks Charles L.
Publication year - 2007
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.062724307
Subject(s) - dihydrofolate reductase , protonation , cofactor , ternary complex , stereochemistry , chemistry , escherichia coli , substrate (aquarium) , loop (graph theory) , conformational change , nicotinamide , biochemistry , enzyme , biology , ion , ecology , mathematics , organic chemistry , combinatorics , gene
We evaluate the pK a of dihydrofolate (H 2 F) at the N 5 position in three ternary complexes with Escherichia coli dihydrofolate reductase (ecDHFR), namely ecDHFR(NADP + :H 2 F) in the closed form (1), and the Michaelis complexes ecDHFR(NADPH:H 2 F) in the closed (2) and occluded (3) forms, by performing free energy perturbation with molecular dynamics simulations (FEP/MD). Our simulations suggest that in the Michaelis complex the pK a is modulated by the Met20 loop fluctuations, providing the largest pK a shift in substates with a “tightly closed” loop conformation; in the “partially closed/open” substates, the pK a is similar to that in the occluded complex. Conducive to the protonation, tightly closing the Met20 loop enhances the interactions of the cofactor and the substrate with the Met20 side chain and aligns the nicotinamide ring of the cofactor coplanar with the pterin ring of the substrate. Overall, the present study favors the hypothesis that N 5 is protonated directly from solution and provides further insights into the mechanism of the substrate protonation.