Study Of Protonation State Of The Glutamic Acid 197/202 In Cholinesterases

Butyrylcholinesterase(BCh E)is one of the important enzymes and is actively involved in the drug discoveries for diverse diseases or health problems.It is one of the important targets of Alzheimer's disease(AD).The situation caused by AD is getting more urgent in that the number of patients is large and growing fast.Tacrine is one of the first cholinesterase inhibitors that was introduced into clinical to treat AD.It targets both BCh E and ACh E,making it a multi-directed ligand.The crystal structure of BCh Etacrine complex(PDB ID 4BDS)shows that there is an unanticipated formyl-proline molecule resolved close to tacrine,raising an essential question on how reliable it is to apply the binding pose revealed in the crystal structure to analyze related experimental observations where no formyl-proline is actually involved.In this study,we answered the above question by performing a series of 100 ns molecular dynamics(MD)simulations,and found the unexpected protonation state of Glu197,which is different from what many researchers thought.We also found the important role of hydrogen bond network and verified the protonation state of Glu197 in other BCh E structures.In Chapter One,we overviewed the research background and theory foundation of this dissertation.In Chapter Two,we studied whether the presence of formyl-proline has an effect on the binding pose of tacrine in BCh E.By performing a series of 100 ns MD simulations,we found that neither simulations without formyl-proline nor with protonated or deprotonated formyl-proline inside the pocket can reveal the same binding pose as in the crystal structure.By analyzing of the structures from the above simulations,we proposed and verified the possibility of Glu197 being protonated.In the simulations with Glu197 protonated,formyl-proline has no effect on the binding pose of tacrine,suggesting one may safely apply the structural information from 4BDS to interpret experimental observations where no formyl-proline is involved.In Chapter Three,we proposed that an important hydrogen bond network associate with a highly conserved water molecule is the key to the understanding of protonation states of Glu197.By careful analysis of simulations in chapter one,we found that a key water can interact with multiple residues including the protonated Glu197,thus forming a hydrogen bond network,stabilizing these residues,and preventing the mobile His438 which is located in a loop region from large conformational change.However,deprotonated Glu197 is unable to form this important hydrogen bond network to stabilize the structure.Furthermore,constant p H MD simulation(Cp HMD)excluded other possibility of the protonation state of Glu197.In Chapter Four,we studied the protonation state of Glu197 in apo BCh E.By performing multiple repeated MD simulations,we proved that the same rule exists in apo BCh E,i.e.,Glu197 should be protonated and the hydrogen bond network is responsible for the local structural stability.In Chapter Five,we studied the protonation state of Glu202(corresponding to Glu197 in BCh E)in the complex of sarin inhibited ACh E and HI6.Crystal structure 2WHP and 5FPP revealed two different binding conformation of HI6,but only the conformation in 5FPP is the effective conformation to reactivate the sarin inhibited ACh E.There is no report of successful simulation that reproduces the effective binding pose of HI6 as in 5FPP.In this study,we first calculated the portion of protonated state of Glu202 in the complex of sarin inhibited ACh E and HI6.Then by comparing the simulations with the protonation state of titrating residues determined by Reduce or constant p H molecular dynamics,we concluded that only with correct protonation state could the simulations reveal the effective binding pose of HI6 as in 5FPP,among which the protonation state of Glu202 plays an important role.This discovery is highly related to the hydrogen bond network stabilized by protonated Glu202.It is worth noting that the Glu197/Glu202 in cholinesterases has been long considered as deprotonated in analyzing experimental observations due to low p Ka of glutamic acid in aqueous solution,in which some interpretations are inconsistent or unclear.Our results provide an important clue that has been long missing for better understanding of these puzzles.In particular,analyzing experimental observations,designing high active inhibitors against cholinesterases,and proposing hypotheses for cholinesterase-catalytic reaction mechanism should have the protonated state of Glu197/Glu202 been considered.