organophosphorus ( op ) compounds are used 
 as pesticides , insecticides , 
 and chemical nerve agents such as sarin ( gb ) , tabun ( ga ) , soman ( gd ) , 
 and vx ( figure 1 ) . op compounds inhibit acetylcholinesterase 
 ( ache ) by forming a covalent adduct with an active site serine residue , 
 leading to overstimulation of the nervous system and subsequently 
 to respiratory failure and death . moreover , covalent adducts of ache undergo spontaneous dealkylation , 
 or  aging  , which renders adduct formation irreversible . therefore , there is great interest in developing new approaches 
 for protection against op compounds . an 
 important detoxification strategy involves using enzymes as catalytic 
 bioscavengers to degrade op compounds before they can reach ache . many enzymes are known 
 to display low - level hydrolase activity 
 toward op compounds and several have been investigated as potential 
 catalytic bioscavengers . substantial effort 
 has been directed toward improving the catalytic activity and enantioselectivity 
 of bioscavengers toward op nerve agents through rational design and 
 directed evolution approaches . notable examples include organophosphorus 
 hydrolase ( oph ) , also called phosphotriesterase ( pte ) , from the soil bacterium brevundimonas diminuta , human serum paraoxonase ( pon1 ) , and diisopropyl fluorophosphatase ( dfpase ) from the european squid , loligo vulgaris . wild - type 
 pon1 and dfpase exhibit a preference for the less toxic ( r)-enantiomers of g - type nerve agents . to design improved variants 
 of op hydrolases , it is beneficial 
 to understand the biochemical mechanisms of these enzymes with various substrates . several aspects of the 
 mechanism of dfp hydrolysis by dfpase have been determined experimentally . for example , the ph  rate profile for the reaction revealed 
 that a protein residue with an apparent pka of 6.8 must be deprotonated for maximal catalytic activity . the experimentally measured kcat for the hydrolysis of dfp by wild - type dfpase is 211 
  9 s. on the 
 basis of mutagenesis studies , active site residue his287 was shown 
 not to play a major role in catalysis and is not essential for activity . single- and multiple - turnover reactions in h2o demonstrated that a carboxylate oxygen from 
 asp229 is incorporated into the dfp hydrolysis product , diisopropylphosphate , establishing that a covalent phosphoenzyme intermediate 
 is formed between asp229 and dfp ( figure 2 ) . 
 a water molecule is then presumed to attack the phosphoacyl enzyme 
 intermediate ( c of asp229 ) to liberate diisopropylphosphate 
 and to complete the catalytic cycle . brnsted analysis of pon1 
 with analogues of paraoxon revealed a lg value of 
 1.6 for leaving groups with pka s > 7 , suggesting that the transition state is very late 
 and leaving group dissociation is fully rate limiting . ( left ) proposed mechanism for phosphoenzyme intermediate 
 formation 
 involving asp229 as the nucleophile . ( i ) asp229 attacks the phosphorus 
 center of dfp to form a pentavalent intermediate and ( ii ) the p  f 
 bond dissociates to form a tetrahedral phosphoenzyme intermediate . ( right ) 
 proposed mechanism for hydrolysis involving an activated water as 
 the nucleophile . ( i ) asp229 abstracts a proton from a water molecule 
 either stepwise or in concert as ( ii ) water attacks the phosphorus 
 center , ( iii ) glu21 abstracts a proton either stepwise or in concert 
 as ( 4 ) water forms a bond with phosphorus , and ( iv ) the p  f 
 bond dissociates . several high - resolution 
 x - ray crystal structures and one joint x - ray / neutron ( x / n ) diffraction 
 structure of dfpase provide insight into 
 the possible binding modes of op substrates and protonation states 
 of key residues and also enable simulation of substrate binding and 
 catalysis . although 
 the amino acid sequences of dfpase and pon1 display significant divergence , each has a six - bladed -propeller fold 
 with two ca metal ions : one required for catalysis and 
 the other , located in the central water tunnel , providing structural 
 integrity . the first -strand of each blade contributes at least one 
 residue to the active site ( figure 3 ) , although 
 they are not all coordinated to the catalytic ca . the x / n 
 structure of dfpase crystallized at ph 6.5 revealed that all active 
 site residues are in their canonical protonation states and that two 
 neutral water molecules , as opposed to hydroxides , are coordinated 
 to the catalytic ca . thus , modeling the nucleophilic 
 attack by asp229 on op compounds does not require examination of any 
 asp229 activation steps by ser271 , although this step has also been 
 proposed as part of the mechanism for dfpase . a recent report of a high - resolution x - ray structure of dfpase determined 
 at 0.85  resolution suggests that a third water molecule , bound 
 to the catalytic ca , shares a proton with asp229 . the authors proposed that this water molecule 
 is partially activated and may play a role in enzyme regeneration . structural 
 depiction of dfpase showing each blade of the six - bladed 
 -propeller fold with the active site residue from each blade 
 shown in the cpk representation . black spheres represent the catalytic 
 ( foreground ) and structural ( background ) ca ions . various aspects of dfpase - catalyzed 
 op hydrolysis mechanisms remain 
 to be determined conclusively . for example , it is not known whether 
 nucleophilic attack on the substrate involves a one - step andn - like transition or 
 proceeds through a trigonal bipyramidal / pentavalent intermediate ( an + dn ) . moreover , the roles of intermolecular interactions in the active 
 site that facilitate elimination of the fluoride leaving group have 
 not been investigated . another question concerns how a water molecule 
 is activated for attack on the tetrahedral phosphoacyl enzyme intermediate . it is also not known whether the hydrolysis of all op compounds share 
 the same mechanism . answers to these questions would be useful in 
 interpreting dfpase and pon1 engineering studies and may help to guide 
 rational enzyme design efforts . here , we perform quantum mechanical / molecular 
 mechanical ( qm / mm ) umbrella sampling simulations with density 
 functional theory 
 to investigate and compare the mechanisms of dfp and ( s)-sarin hydrolysis by dfpase . we then analyze the underlying energetics 
 of nucleophilic attack by a carboxylate nucleophile on both substrates 
 and characterize important differences between them . lastly , we discuss 
 the implications of our findings for designing improved nerve agent 
 bioscavengers . although the substrates diisopropyl 
 fluorophosphate ( dfp ) and isopropyl methylphosphonofluoridate ( sarin ) 
 were included in the qm subsystem during the qm / mm simulations , we 
 performed initial system equilibration steps with classical mm md . thus , we generated charmm force field parameters for dfp and sarin 
 by analogy to existing parameters and made appropriate modifications . the paramchem web server ( www.paramchem.org ) was used to generate initial topology and 
 parameter sets for diisopropylphosphate and isopropyl methylphosphonate , 
 which were then modified for use with dfp and sarin . equilibrium bond 
 lengths and angles were obtained from b3lyp/6 - 31+g(d ) geometry optimizations , 
 and charges were obtained from mulliken population analysis with orca . atomic 
 coordinates for the simulations were obtained from the 1.73  
 resolution x - ray cocrystal structure of dfpase from l. vulgaris with the dfp analogue dicyclopentyl phosphoramidate 
 ( dcppa ) ( pdb entry 2gvv ) . protonation states were assigned 
 on the basis of a joint x - ray / neutron diffraction structure of apo dfpase ( pdb entry 3byc ) , which 
 revealed that all protein side chains are in their standard ph 7 ionization 
 states . two systems were simulated : one with dfp as the substrate 
 and the other with ( s)-sarin . dcppa was modified 
 to generate dfp by replacing the amidate group with fluoride and the 
 two o - cyclopentyl groups with o - isopropyls . because dcppa binds in 
 an unreactive orientation and because it is well - established that 
 the substrate must be aligned for in - line attack on phosphorus by asp229 , the bound substrate models were rotated 
 120 clockwise about the ca - coordinating 
 o  p bond ( supporting information , figures 
 s1 and s2 ) . this modification produced a reactive orientation 
 while also preserving ca coordination to the substrate . the michaelis complex of dfpase with ( s)-sarin was 
 constructed by replacing the appropriate o - isopropyl 
 of dfp with a methyl group . crystallographic water molecules 
 were retained , and each system was fully solvated in an orthorhombic 
 box of water molecules with a minimum distance of 11  from the 
 protein to the nearest face of the box . five na cations 
 were added with the autoionize plugin of vmd to neutralize the charge of each system . both systems contained 
 40  000 atoms . the charmm27 force field with cmap corrections and 
 the tip3p water model were used to describe 
 the protein and solvent , respectively . one thousand steps of energy 
 minimization were performed using the steepest descent algorithm . periodic boundary conditions were applied , and the particle mesh ewald 
 method ( 1 grid point  ) was used to describe long - range electrostatic effects . the shake algorithm was used to constrain 
 all bonds to hydrogen , enabling the use of a 2 fs time step . molecular 
 dynamics ( md ) simulations were initiated by heating the systems to 
 300 k over 30 ps and rescaling the velocities every 1000 steps . a 
 5.0 kcal mol  harmonic 
 restraint potential was imposed on all protein and substrate heavy 
 atoms during the heating stages . lastly , 0.5 ns md simulations were 
 performed in an nvt ensemble to equilibrate each system and to obtain 
 initial configurations for qm / mm simulations . a 1.0 kcal mol harmonic restraint potential was imposed 
 on all protein and substrate heavy atoms during this equilibration 
 stage . representative structures were 
 extracted near the end of the classical md simulations and used as 
 initial configurations . all water molecules with an oxygen atom greater 
 than 30  from the phosphorus atom of the substrate were deleted 
 to create a spherical boundary model of the system , a common approach 
 that has been shown to yield good accuracy in investigations of enzymatic 
 reactions . the qm region included 
 the dfp or ( s)-sarin substrate , the side chains of 
 glu21 , asn120 , asn175 , asp229 , ser271 , and asn272 , the catalytic ca , and two crystallographic water molecules coordinated to 
 ca . hydrogen link atoms were used to describe the qm / mm 
 boundary , and these were automatically placed 1.0  away from 
 c along the cc bond axis for side chains 
 in the qm region by pdynamo . the full qm regions contained 82 and 
 75 atoms for dfp and ( s)-sarin , respectively . the 
 side chain of asp121 was also included in the qm region during the 
 umbrella sampling ( us ) simulations of the hydrolysis of the phosphoenzyme 
 intermediate with dfp substrate , for a total of 88 qm atoms . all water 
 molecules and protein residues containing an atom greater than 20 
  from the phosphorus atom of the substrate were held fixed . for the us simulations , the qm region was described with the gradient - corrected 
 bp86 functional with an ahlrichs split - valence basis set augmented 
 with polarization and pople diffuse functions on all n , o , f , and 
 p atoms . the resolution - of - the - identity approximation 
 with the svp / j auxiliary basis set was 
 used to reduce the computational cost of integral evaluation . in addition , 
 grimme atom - pairwise empirical dispersion corrections ( i.e. , d3 ) were included . hereafter , we refer to the combination of this dft 
 approach and the charmm molecular mechanics potential as dft / mm . initial structures for the us simulations 
 were obtained by performing a series of constrained geometry optimizations 
 along a reaction coordinate defined as the mass - weighted distance 
 difference between o(asp229)p and p  f ( referred 
 to hereafter as the andn reaction coordinate ) . the us simulations were divided into 
 48 windows , with each having a harmonic restraint potential of 475 
 kcal mol  . a 25 ps equilibration 
 ( 1 fs time step ) was first performed for each window with the semiempirical 
 pm6/mm hybrid potential . each window 
 was then further equilibrated with the dft / mm potential for 3 ps , 
 during which time the velocities were scaled every 100 steps until 
 a temperature of 300 k was reached . each window was then sampled for 
 20 ps in an nvt ensemble , and the reaction coordinate value was recorded 
 at every time step . free energy profiles were reconstructed with the 
 weighted histogram analysis method ( wham ) , as implemented in the program wham , version 2.0.9 . initial geometries 
 for us simulations of the hydrolysis of the dfp phosphoenzyme intermediate , 
 in which an activated water attacks c of asp229 , were obtained 
 from dft / mm potential energy scans ( see below ) in which a hydroxide 
 was formed by transferring a proton from a water molecule to asp121 . this water molecule is coordinated to the catalytic ca and is in close proximity to asp121 in the joint x - ray / neutron structure 
 ( pdb entry 3byc ) . the reaction coordinate for these 
 us simulations was defined as the distance difference between c(asp229)o(asp229 ) 
 and ohydroxide c(asp229 ) , and 21 us windows 
 were used to construct the potential of mean force . the fluoride anion 
 was removed from the tetrahedral phosphoenzyme model for the simulations 
 of subsequent hydrolysis steps . dft / mm potential energy ( pe ) 
 scans were performed to characterize proton transfer energetics in 
 the tetrahedral phosphoenzyme intermediate . the reaction coordinate 
 was defined as the distance difference between owater  h 
 and o(asp121)h . gas - phase geometry optimizations were 
 performed for acetate  dfp and acetate(s)-sarin complexes , representative of pentavalent asp229substrate 
 complexes , with the bp86,mpwpw91,mpw1pw91 , and b3lyp functionals and the 6 - 31+g(d ) basis set with orca . relaxed potential energy scans were then 
 carried out by varying the acetate - o  p(dfp / sarin ) distance 
 from 1.85 to 3.05  . after classical 
 md equilibration and subsequent qm / mm optimization of preliminary 
 minimum energy paths obtained with the reaction coordinate driving 
 method , dft / mm us simulations were carried out to determine free energy 
 profiles for each step of the reaction . the simulations indicate that 
 the reaction proceeds by a two - step addition  elimination ( an + dn ) mechanism , 
 passing through a pentavalent intermediate common in phosphoryl transfer 
 reactions ( figure 4 ) . a shallow free energy basin is present for the michaelis complex 
 ( reaction coordinate  0.8  , see the methods section ) . in the michaelis complex , the 
 phosphoryl oxygen of dfp and the catalytic ca are in 
 close contact ( 2.6  ) and remain in similar proximity 
 throughout all us simulations . the pro - so - isopropyl group of dfp forms contacts with a dfpase surface 
 comprising the side chain of arg146 and the hydrophobic side chains 
 of met148 , phe173 , and thr195 , whereas the opposite face of this group 
 is exposed to solvent . the pro - ro - isopropyl group of dfp forms contacts with glu21 , trp244 , ser271 , 
 and his287 . the pro - r group has less contact with 
 water because of shielding by the other o - isopropyl 
 group . the fluorine of dfp is hydrogen - bonded to one water molecule 
 and lacks any direct interactions with the enzyme in the michaelis 
 complex . ( a ) dft / mm us free energy profile for the an + dn reaction of dfpase with dfp 
 substrate . the reaction coordinate is defined as the mass - weighted 
 distance difference between o(asp229)p and p  f 
 and progresses from the michaelis complex though the first transition 
 state ( ts-1 ) to the pentavalent phosphoenzyme intermediate and then 
 through the second transition state ( ts-2 ) to the tetrahedral phosphoenzyme 
 intermediate at a reaction coordinate value beyond 1.4 . statistical 
 errors range from 0.01 kcal mol near the michaelis 
 complex to 0.05 kcal mol near the tetrahedral 
 phosphoenzyme intermediate . ( d ) representative snapshot of the rate - limiting transition 
 state in which fluoride dissociates from the pentavalent intermediate . note that ca - coordinating residues asn120 and asn175 
 are omitted in panels b  d for clarity . the computed free energy barrier for the nucleophilic addition 
 step to form a metastable pentavalent species is 8.5 kcal mol , and this step is endergonic by 6.7 kcal mol ( figure 4 ) . at ts - i , an additional 
 water f(dfp ) hydrogen bond is formed that is not present earlier 
 in the reaction . this water molecule is only weakly associated with 
 the catalytic ca , with an average oxygen ca distance of 3.75  ( figure 4 ) , but forms a hydrogen bond with the side chain of asn120 . the pentavalent 
 intermediate differs from ts-1 primarily in that the water molecule 
 that was weakly coordinated to ca and hydrogen - bonded 
 to the fluorine of dfp is now more strongly coordinated to ca , with an average oxygen a hydrogen bond between one of the amide hydrogens 
 of the asn175 side chain and the phosphoryl oxygen of the substrate 
 also forms and remains intact for the remainder of the simulations . the free energy barrier for the fluoride elimination step relative 
 to the pentavalent intermediate with dfp substrate was computed to 
 be 7.5 kcal mol , yielding an overall free energy 
 barrier of 14.2 kcal mol . fluoride elimination 
 was found to proceed through a late transition state ( figure 4 ) , in agreement with a brnsted analysis of 
 pon1 with paraoxon analogues as substrates . at the transition state , with p  f separations averaging 2.3 
  , these two centers are not yet solvent - separated and maintain 
 a direct path for recombination . the transition - state structures for 
 fluoride elimination reveal an additional water molecule interacting 
 with the departing fluoride . asn120 has switched from hydrogen bonding 
 to fluorine through a water molecule to a direct hydrogen bond , which 
 is retained for the rest of the simulations up through the formation 
 of the tetrahedral phosphoenzyme species . to clarify further , the 
 fluorine of dfp has four hydrogen - bonding partners at ts-2 , with three 
 water molecules and the side chain of asn120 all serving as donors . the underlying physical basis of this late transition state stems 
 partly from the requirement of the departing fluoride leaving group 
 to reach a favorable solvation environment for the free energy to 
 reach a minimum . it is also worth noting that the water molecules 
 partially solvating the nascent fluoride at ts-2 are adjacent to hydrophobic 
 surfaces . as a result , the overall reaction free energy for 
 the reaction 
 is less certain . even when the andn reaction coordinate corresponded to the 
 tetrahedral phosphoacyl enzyme intermediate ( i.e. , with an average 
 p  f distance of 4  ) , no free energy minimum was 
 found for the range of reaction coordinate values considered ( figure 4 ) . a reaction occurring at or near the solvent - exposed 
 surface of an enzyme with a product or leaving group approaching bulk 
 solvent would not be expected to show a free energy minimum for the 
 product state until that species is completely solvated , as has been 
 shown in similar studies . in addition , 
 whether a free energy minimum is located at larger p  f separations 
 is of minimal concern here because the aim of our study is to identify 
 the factors that contribute to the overall free energy barrier . thus , 
 our conclusions are not affected by neglecting to identify a free 
 energy minimum for this step . the last 
 step of the catalytic cycle for op hydrolysis by dfpase has not been 
 characterized conclusively by experiments because it is not rate limiting . nevertheless , dft / mm simulations suggest a possible mechanism in which 
 a water molecule coordinated to ca performs a nucleophilic 
 attack on the tetrahedral intermediate . in the joint x - ray / neutron 
 structure of dfpase and in the present 
 simulations , a water molecule is coordinated to the catalytic ca and is also hydrogen - bonded to the side chain of asp121 . dft / mm pe scans indicate that in both the michaelis complex and in 
 the pentavalent intermediate , this water molecule and asp121 are in 
 strong hydrogen - bonding contact ( hydrogen - acceptor distance = 1.49 
  , donor  hydrogen - acceptor angle = 172 ) in the minimum 
 energy structures ( supporting information , figure 
 s4 ) . this geometry is consistent with the assignment of two 
 protons on this water molecule from neutron diffraction . one of the main differences between the 
 michaelis complex , the pentavalent phosphoenzyme intermediate , and 
 the tetrahedral phosphoacyl enzyme intermediate is in the coordination 
 to the catalytic ca ion . progressing from the michaelis 
 complex to the pentavalent intermediate , the oxygens of asn120 , the 
 phosphoryl oxygen of dfp , and the water molecule that is hydrogen - bonded 
 to asp121 move closer to the catalytic ca , whereas the 
 other side chains move further away . in the phosphoacyl enzyme intermediate , ca distances are longer than in the michaelis complex and the pentavalent 
 intermediate except for those involving the two coordinating water 
 molecules ( supporting information , figure s3 ) . interestingly , by taking a representative tetrahedral phosphoenzyme 
 intermediate snapshot from the us simulations and including asp121 
 in the qm region , we found that a proton on the water molecule was 
 transferred spontaneously to asp121 ( supporting 
 information , figures s3 and s4 ) . dft / mm us simulations 
 of the hydrolysis of the tetrahedral phosphoenzyme 
 intermediate indicate that the reaction has a barrier of only 1.3 
 kcal mol and is endergonic by 6.9 kcal mol ( figure 5 ) . the small free 
 energy barrier is consistent with experiments showing that this step 
 is not rate limiting . as hydroxide attacks 
 the phosphoacyl enzyme intermediate , the geometry around c 
 becomes tetrahedral and the co bond begins to 
 break as the geometry around c becomes planar . during the reaction , 
 a hydrogen bond forms between the hydroxyl group of ser271 and the 
 remaining o(asp229 ) . the distance between the phosphoryl oxygen 
 and ca decreases , consistent with the computed increase 
 in partial charge on the phosphoryl oxygen as the reaction proceeds . ( a ) dft / mm 
 us free energy profile for the nucleophilic attack of 
 an activated water on the phosphoenzyme intermediate . ( b ) snapshot 
 showing the interactions that stabilize hydroxide , which is separated 
 from c of asp229 by 3.1  . note that ca - coordinating residue asn120 is omitted in panels b and c for clarity . the experimentally determined 
 free energy barrier , obtained from kcat with transition state theory , is 14.3 kcal 
 mol . our simulations 
 yield an overall reaction free energy of 14.2 kcal mol and are consistent with other available experimental kinetics and 
 mutagenesis data , suggesting that the computationally determined mechanism 
 is reasonable . the mechanism for dfpase - catalyzed inactivation of 
 dfp was found to proceed through the nucleophilic attack by asp229 
 on the phosphorus center of dfp to form a transient pentavalent intermediate 
 followed by p f dissociation to yield a covalent phosphoacyl 
 intermediate in the rate - limiting step . attack on c of asp229 
 in the phosphoacyl intermediate by an activated water molecule then 
 yields diisopropylphosphate to complete the catalytic cycle . the kcat / km value for 
 dfpase with ( s)-sarin was determined to be 4.2  
 10 m s , but kcat and km were not determined independently of each 
 other . however , assuming that both ( s)-sarin and 
 dfp have similar binding affinities to dfpase , the free energy barrier 
 derived from kcat for ( s)-sarin hydrolysis would be expected to be similar to that of dfp . 
 surprisingly , the computed free energy barrier for ( s)-sarin hydrolysis by the same mechanism as dfp was significantly 
 higher . for ( s)-sarin , the pentavalent intermediate 
 is completely absent and the overall free energy barrier is > 14 
 kcal 
 mol higher than for dfp , at 28.6 kcal mol ( figure 6 ) . as is the case 
 for dfp , the transition state for the reaction is very late , with 
 an o(asp229)p distance of 1.75  and a 
 p the formation of a pentavalent 
 species with asp229(s)-sarin requires 16 
 kcal mol more than the analogous species in dfp . 
 progressing from this point in the reaction coordinate ( rc  
 0.10  ) to the ts requires an additional 56 kcal mol for ( s)-sarin compared to 7.6 kcal 
 mol for dfp . thus , the major difference between 
 these reactions is the energetic cost of forming a pentavalent enzyme the computed high free energy barrier for ( s)-sarin hydrolysis by this pathway is clearly inconsistent with experimental 
 kinetics data , as dfpase hydrolyzes ( s)-sarin almost 
 as efficiently as it does dfp ( kcat / km = 4.2  10 versus 5.6  
 10 m s ) . therefore , the present findings indicate that 
 the hydrolysis mechanism for ( s)-sarin is very likely 
 to be different from that of dfp . ( left ) dft / mm us free energy profile for 
 the andn reaction 
 of dfpase with ( s)-sarin . the arrow from the dfp : dfpase 
 pentavalent intermediate 
 basin to the corresponding point in the dfpase:(s)-sarin free energy profile is shown to highlight the complete lack 
 of this intermediate in the reaction with ( s)-sarin . statistical errors range from 0.01 kcal mol near 
 the michaelis complex to 0.06 kcal mol near the 
 tetrahedral phosphoenzyme intermediate . ( right ) representative snapshot 
 of the transition state from the dft / mm us simulation of the dfpase:(s)-sarin reaction . in the michaelis complex , one water molecule is hydrogen - bonded 
 to the fluorine of sarin , and one of the amide hydrogens from the 
 side chain of asn120 also makes frequent hydrogen - bonding contacts 
 with fluorine . as observed in the dfpase : dfp simulations , the phosphoryl 
 oxygen of ( s)-sarin remains in close contact ( 2.6 
  ) with the catalytic ca throughout the us simulations . one major difference between the dfp and ( s)-sarin 
 michaelis complexes is that with ( s)-sarin o 
 from asp229 is hydrogen - bonded to two water molecules . one of these 
 water molecules is also positioned to perform an in - line nucleophilic 
 attack on phosphorus of dfp ( figure 7 ) . this 
 water molecule is displaced during the formation of the pentavalent 
 structure of dfpase:(s)-sarin , but the other remains 
 stably bound in the active site . the hydrogen - bonding partners with 
 fluorine remain the same as in the michaelis complex . at the transition 
 state , the water molecule interacting with o of asp229 becomes 
 hydrogen bonded to o - isopropyl oxygen late in the 
 reaction . in the final us window , the fluoride is hydrogen - bonded 
 to four water molecules . as with the departing fluoride of dfp , the 
 water molecules that are hydrogen - bonded to the fluoride of ( s)-sarin are adjacent to a hydrophobic surface of the enzyme 
 and are not in a favorable solvation environment . the hydrogen bond 
 between asn120 and the fluoride has been replaced by another hydrogen 
 bond between asn120 and the phosphoryl oxygen of the phosphoenzyme 
 intermediate . snapshot from dft / mm simulations of dfpase/(s)-sarin 
 supporting a nucleophilic attack on the phosphorus center of ( s)-sarin hydrolysis by a water molecule upon activation 
 by proton transfer to asp229 . ca - coordinating residues asn120 and asn175 
 are omitted for clarity . to investigate the underlying reactivity of asp229 as a nucleophile 
 reacting with dfp and ( s)-sarin , we performed gas - phase 
 geometry optimizations and pe scans with the bp86 , b3lyp , mpwpw91 , and mpw1pw91 density functionals 
 and the 6 - 31+g(d ) basis set on isolated models of asp229substrate 
 adducts , with asp229 being represented by acetate . electron - donating substituents at equatorial positions are 
 known to destabilize pentavalent phosphorus species . dfp is better able to stabilize the negative charge from 
 the carboxylate nucleophile with its two o - isopropyl 
 substituents than ( s)-sarin , which contains one o - isopropyl and one methyl group . from the dft / mm simulations , 
 the larger partial negative charge on the nucleophilic asp229o 
 in ( s)-sarin versus dfp and the large partial negative 
 charge on the methyl carbon in ( s)-sarin support 
 the above rationale ( supporting information , table 
 s1 ) . p 
 distance in ( left ) acetate  dfp and ( right ) acetate sarin 
 obtained with the bp86 , b3lyp , mpwpw91 , and mpw1pw91 functionals and the 6 - 31+g(d ) basis set . the physicochemical factors that 
 lead to the vastly different free 
 energy profiles for the two substrates stem from ( 1 ) the difference 
 in the ability of the two substrates to accommodate the additional 
 negative charge from the incoming asp229 nucleophile and ( 2 ) competing 
 interactions between the electrophilic phosphorus and water molecules 
 with asp229 . thus , the findings from the qm / mm simulations can be 
 traced to differences in the electrophilicity and reactivity of the 
 phosphorus centers in dfp and ( s)-sarin . given 
 the high calculated free energy barrier for the concerted 
 but highly asynchronous ( andn ) reaction of asp229 with ( s)-sarin 
 and the complete absence of a metastable pentavalent intermediate , 
 we considered the possibility of an alternative mechanism in which 
 asp229 activates an ordered water molecule , which then attacks phosphorus 
 ( either in a stepwise or concerted reaction ) . in the first five windows 
 of the us simulations of dfpase:(s)-sarin , a water 
 molecule from the mm region is hydrogen - bonded to both asp229 and 
 ser271 and is positioned between these residues and ( s)-sarin ( figure 7 ) . the catalytic ca is not expected to affect the nucleophilicity of this water molecule 
 because it is 5  away in the simulations . inclusion 
 of this water molecule in the qm region would be expected to polarize 
 it further through its hydrogen - bonding interactions with asp229 . thus , it is possible that asp229 could activate the water molecule 
 for nucleophilic attack on the substrate rather than performing the 
 nucleophilic attack directly . simulating this process would require 
 exploration of 2d free energy surfaces , the computational cost of 
 which is prohibitive with the dft / mm methods used here . whereas only 
 multiple - turnover kinetics assays with dfp substrate in h2o yielded an o - labeled hydrolysis product , our simulations predict that both single- and 
 multiple - turnover assays with ( s)-sarin should incorporate 
 the o label into the hydrolysis product , isopropyl methylphosphonate . a bioinformatics study of six - bladed -propeller enzymes that carry out nucleophilic 
 attack placed dfpases within a subgroup consisting of smp-30/gluconolactonase / luciferin - regenerating 
 ( sgl ) enzymes , whereas the paraoxonases were placed in an arylesterase 
 subgroup . smp-30 is involved in l - ascorbic acid biosynthesis 
 in nonprimate mammals but has also been investigated for its op hydrolytic 
 capabilities . smp-30 shares some active 
 site residues with dfpase but has very poor activity against g - type 
 nerve agents due , in part , to its poor substrate binding characteristics . the promiscuity of enzymes bearing the six - bladed 
 -propeller fold is immense , thus 
 seemingly providing an excellent platform to design enzymes with alternate 
 functions . indeed , dfpase has even served as a scaffold to design 
 a diels  alderase . on the 
 basis of in vivo protection experiments , catalytic efficiencies ( kcat / km )  
 10 m min are required 
 for effective prophylaxis against 2  ld50 of g - type 
 agents using minimal enzyme doses ( 50 mg/70 kg ) . through a combination of rational design and 
 directed evolution , pon1 variants have been developed with rates of 
 hydrolysis of g - type nerve agents that are enhanced by at least 340-fold 
 relative to wild type . the catalytic efficiencies ( kcat / km ) of the best pon1 variants 
 with gb , gd , and gf substrates are 5  10 , 1.2  
 10 , and 2.9  10 m s , respectively . a similar approach combining 
 rational design and directed evolution was used to engineer pte variants 
 that hydrolyze the ( s)-enantiomers of gb , gd , and 
 gf with kcat / km values of 2  10 , 5  10 , and 
 8  10 m s , respectively . wild - type dfpase exhibits a catalytic efficiency 
 for ( s)-gb ( kcat / km = 4.2  10 m s ) that is almost 1 order of magnitude higher 
 than the current best pon1 variant . for ( s)-gf ( kcat / km = 1.7  
 10 m s ) , dfpase 
 has 17-fold lower activity than the best pon1 variant . a rationally designed quadruple mutant ( glu37ala / tyr144ala / arg146ala / thr195met ) 
 of dfpase displayed reversed enantioselectivity to favor the more 
 toxic ( s)-enantiomers of gb and gf , simultaneously 
 increasing the catalytic efficiencies to 2.3  10 and 4.9  10 m s , respectively . exchanging three bulky 
 residues with alanines resulted in a more accessible active site , 
 and introducing a negative design element ( thr195met ) hindered the 
 binding of the less - toxic ( r)-enantiomers of gb and 
 gf . the qm / mm simulations performed in this work have implications 
 for engineering dfpase nerve agent bioscavengers and possibly other 
 six - bladed -propeller enzymes such as pon1 and smp-30 . the simulations 
 suggest ways to optimize enzymes for more efficient catalysis . to 
 lower the barrier for fluoride elimination , the local environment 
 should be sufficiently hydrophilic to allow for fluoride solvation . 
 in dfpase , hydrophobic residues such as met90 , met148 , and phe173 
 ( supporting information , figure s2 ) create 
 an unfavorable solvation environment for the fluoride anion and may 
 hinder the expulsion of the leaving group . therefore , proper placement 
 of positively charged side chains ( arg and lys ) or hydrogen - bond donors 
 that could interact favorably with the developing negative charge 
 on fluorine or facilitate improved solvation is a strategy worth exploring . 
 our simulations also show that tyr144 is near the path of the departing 
 fluoride anion in the rate - limiting elimination step . tyr144 was mutated 
 to ala in the quadruple mutant of dfpase , which presumably allows greater solvation of fluoride and lowers 
 the free energy barrier for the rate - determining step . furthermore , 
 the present simulations also provide an interpretation 
 of the effect of the mutations reported by goldsmith et al . in their 
 rational and directed evolution design efforts to develop a broad - spectrum 
 g - type nerve agent detoxifying enzyme . in repon1 variant iig1 , active site residues thr332 and his115 were 
 mutated to ser and ala , respectively , thereby creating an active site 
 that was almost identical to that of dfpase . the second - sphere his134arg 
 mutation was thought to compensate for the his115ala mutation . however , 
 because his134 is oriented toward the active site in pon1 structures , 
 mutating it to arg while also changing his115 to ala might expose 
 the positively charged arg side chain to the active site and place 
 it in a position to assist in the departure of the fluoride leaving 
 group . recall that this reaction has a very late transition state 
 ( i.e. thus , the favorable interactions between an exposed arg134 
 and the leaving group may partially explain the large increase in 
 catalytic efficiency seen in the pg11 variant for soman ( gd ) hydrolysis . asp136 , like 
 his134 , is oriented toward the active site and would not interact 
 favorably with a departing fluoride anion . thus , its replacement could 
 assist in lowering the barrier for the elimination reaction , although 
 a similar mutation in dfpase would not be possible because the aspartate 
 is located in a nonhomologous loop region . mechanistic interpretations 
 for dfpase do not necessarily transfer 
 to pon1 because their active sites differ slightly , most notably with 
 respect to his115(pon1)/ala74(dfpase ) , which enables other mechanistic 
 possibilities to be entertained . for example , instead of a direct 
 nucleophilic attack by asp on the substrate , it has been proposed 
 that a his134his115 dyad in pon1 activates a water molecule 
 for attack . extensive site - directed mutagenesis 
 studies support this mechanism for the pon1-catalyzed hydrolysis of 
 lactones , but these residues do not appear to play roles in op hydrolase 
 activity . on the basis of md simulations , 
 asp269 in pon1 has also been proposed to activate a water molecule 
 for attack on op substrates . perhaps the most 
 significant implication of the present findings 
 is that replacing an electron - acceptor group ( o - isopropyl ) 
 in the dfp substrate with an electron donor ( methyl ) in ( s)-sarin is sufficient to alter the hydrolysis reaction mechanism . this result , determined with quantitatively accurate dft / mm simulations , 
 was unexpected given that the catalytic efficiencies for both substrates 
 are similar . the methyl group in sarin precludes the formation of 
 a pentavalent phosphoenzyme intermediate , as we have shown with simple 
 gas - phase calculations on acetate  sarin and acetate of course , the enzyme environment can also be a significant 
 factor in stabilizing a pentavalent phosphorus structure , but the calculations indicate that the enzyme 
 environment actually contributes further to the destabilization with 
 ( s)-sarin . the less bulky methyl group allows water 
 molecules to be in closer proximity to asp229 , and the interactions 
 of water with asp229 are likely to be more favorable than those with 
 phosphorus . other nerve agents such as soman and vx also have a single 
 methyl group bonded to phosphorus . these nerve agents may also undergo 
 hydrolysis by dfpase and related enzymes through an alternate mechanism , 
 although other factors will undoubtedly play a role . the reactivity 
 of carboxylate nucleophiles toward a given op substrate 
 should be considered in bioscavenger design efforts . however , it remains 
 to be seen whether nucleophilic attack by asp229 or water / hydroxide 
 is preferable . nevertheless , insight gained from mechanistic simulation 
 studies can serve as a guide for rational design and as a starting 
 point for further optimization and refinement through directed evolution .
organophosphorus 
 ( op ) nerve agents such as ( s)-sarin 
 are among the most highly toxic compounds that have been synthesized . 
 engineering enzymes that catalyze the hydrolysis of nerve agents (  bioscavengers  ) 
 is an emerging prophylactic approach to diminish their toxic effects . 
 although its native function is not known , diisopropyl fluorophosphatase 
 ( dfpase ) from loligo vulgaris catalyzes 
 the hydrolysis of op compounds . here 
 , we investigate the mechanisms 
 of diisopropylfluorophosphate ( dfp ) and ( s)-sarin 
 hydrolysis by dfpase with quantum mechanical / molecular mechanical 
 umbrella sampling simulations . 
 we find that the mechanism for hydrolysis 
 of dfp involves nucleophilic attack by asp229 on phosphorus to form 
 a pentavalent intermediate . 
 p 
 f bond dissociation then yields 
 a phosphoacyl enzyme intermediate in the rate - limiting step . 
 the simulations 
 suggest that a water molecule , coordinated to the catalytic ca2 + , donates a proton to asp121 and then attacks the tetrahedral 
 phosphoacyl intermediate to liberate the diisopropylphosphate product . 
 in contrast , the calculated free energy barrier for hydrolysis of 
 ( s)-sarin by the same mechanism is highly unfavorable , 
 primarily because of the instability of the pentavalent phosphoenzyme 
 species . 
 instead , simulations suggest that hydrolysis of ( s)-sarin proceeds by a mechanism in which asp229 could activate 
 an intervening water molecule for nucleophilic attack on the substrate 
 . 
 these findings may lead to improved strategies for engineering dfpase 
 and related six - bladed -propeller folds for more efficient 
 degradation of op compounds .