TY - JOUR
T1 - The role of the Met20 loop in the hydride transfer in Escherichia coli dihydrofolate reductase
AU - Mhashal, Anil R.
AU - Vardi-Kilshtain, Alexandra
AU - Kohen, Amnon
AU - Major, Dan Thomas
N1 - Publisher Copyright: © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2017/8/25
Y1 - 2017/8/25
N2 - A key question concerning the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) is whether the met20 loop is dynamically coupled to the chemical step during catalysis. A more basic, yet unanswered question is whether the met20 loop adopts a closed conformation during the chemical hydride transfer step. To examine the most likely conformation of the met20 loop during the chemical step, we studied the hydride transfer in wild type (WT) ecDHFR using hybrid quantum mechanics-molecular mechanics free energy simulations with the met20 loop in a closed and disordered conformation. Additionally, we investigated three mutant forms (I14X; X=Val, Ala, Gly) of the enzyme that have increased active site flexibility and donor-acceptor distance dynamics in closed and disordered met20 loop states. We found that the conformation of the met20 loop has a dramatic effect on the ordering of active site hydration, although the met20 loop conformation only has a moderate effect on the hydride transfer rate and donor-acceptor distance dynamics. Finally, we evaluated the pKa of the substrateN5position in closed and disordered met20 loop states and found a strong correlation between N5 basicity and the conformation of the met20 loop.
AB - A key question concerning the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) is whether the met20 loop is dynamically coupled to the chemical step during catalysis. A more basic, yet unanswered question is whether the met20 loop adopts a closed conformation during the chemical hydride transfer step. To examine the most likely conformation of the met20 loop during the chemical step, we studied the hydride transfer in wild type (WT) ecDHFR using hybrid quantum mechanics-molecular mechanics free energy simulations with the met20 loop in a closed and disordered conformation. Additionally, we investigated three mutant forms (I14X; X=Val, Ala, Gly) of the enzyme that have increased active site flexibility and donor-acceptor distance dynamics in closed and disordered met20 loop states. We found that the conformation of the met20 loop has a dramatic effect on the ordering of active site hydration, although the met20 loop conformation only has a moderate effect on the hydride transfer rate and donor-acceptor distance dynamics. Finally, we evaluated the pKa of the substrateN5position in closed and disordered met20 loop states and found a strong correlation between N5 basicity and the conformation of the met20 loop.
UR - http://www.scopus.com/inward/record.url?scp=85028389218&partnerID=8YFLogxK
U2 - https://doi.org/10.1074/jbc.M117.777136
DO - https://doi.org/10.1074/jbc.M117.777136
M3 - مقالة
C2 - 28620051
SN - 0021-9258
VL - 292
SP - 14229
EP - 14239
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 34
ER -