the chemical reactions 
 that make life possible are dependent on 
 enzymes for their regulation . therefore , to understand how enzyme 
 function evolves is to understand how life evolves at the most fundamental 
 molecular level .
it has become increasingly clear that the catalytic 
 promiscuity of many enzymes allows them to acquire new functions on 
 time scales much faster than would be expected from the typical time 
 scales for divergence of new enzyme functions by natural evolution .
that is , the promiscuous side activities provide 
 an evolutionary starting point for these enzymes , as they allow new 
 activities to evolve without the selective pressure that is being 
 placed on the native reaction .
this in turn acts as an evolutionary 
  trampoline  that allows a more rapid response to environmental 
 change . in the present work ,
we have used the catalytically promiscuous 
 organophosphatase serum paraoxonase 1 ( pon1 ) as a model system to understand the structural origins 
 of the selectivity and promiscuity of this enzyme and have then performed 
 comparisons to other structurally diverse organophosphatases in order 
 to find the chemical commonalities that facilitate their organophosphatase 
 activities . in particular , as many organophosphatases have evolved 
 from enzymes that were originally lactonases and/or arylesterases 
 ( and retain these activities to varying degrees ) , we have focused on the particular features necessary to facilitate 
 lactone and ester hydrolysis that are common among these enzymes .
pon1 is a calcium - dependent hydrolytic enzyme that is found in 
 all mammalian species . in vivo , this 
 enzyme forms complexes with the membrane - like surface of high - density 
 lipoprotein ( hdl ) and contributes to hdl s antioxidant properties . as with many other organophosphatases , structure 
activity 
 studies suggest that the native activity of pon1 is its lactonase 
 activity , with a particular preference for - 
 and -lactones with long alkyl side chains as its primary substrates . 

the enzyme is broadly promiscuous , however , and can catalyze the hydrolysis 
 of multiple substrates , including not just lactones and thiolactones 
 but also esters , organophosphates , and 
 carbonates .
pon1 has been extensively 
 studied from a biological and biomedical point of view , primarily 
 because of its role in atherosclerosis .
in addition 
 to its direct biomedical relevance , pon1 also shows great promise 
 as a biotherapeutic because of its ability to hydrolyze a broad range 
 of organophosphates , including pesticides and nerve agents such as 
 sarin , soman , and vx .
structurally , pon1 is six - bladed 
 -propeller with a central 
 tunnel containing two calcium ions approximately 7.4  apart 
 ( figure 1a ) . of these calcium ions ,
the calcium ion buried 
 further into the tunnel is structural and essential for pon1 s 
 conformational stability , whereas the second solvent - exposed calcium 
 ion residing at the bottom of the active site cavity is catalytic , 
 playing a role both in substrate positioning and in activating the 
 p(c)=o ester bonds of the substrate ( figure 1b ) . at the 
 top of the active site
are three helices , highlighted in salmon in figure 1a , of which the first 
 two ( helices h1 and h2 ) are believed to play a role in mediating pon1hdl 
 interactions on the basis of both structural analysis and selective 
 mutagenesis . finally , an active site loop 
 comprising residues 7081 , which is absent in all apo crystal 
 structures of pon1 because of its high flexibility , closes over the 
 active site upon binding of a ligand ( 2-hydroxyquinoline , figure 1a ) .
( a ) overall structure 
 of repon1 in complex with the inhibitor 2-hydroxyquinoline 
 ( 2hq ) obtained at ph 6.5 ( pdb i d 3srg ) . here the 
 flexible loop ( residues 7081 ) is highlighted in red , the three 
 surface helices are highlighted in salmon , the ca ions 
 are highlighted in green , and 2hq is highlighted in orange .
( b ) close - up 
 of the active site tunnel leading from the catalytic ca to the surface helix h2 .
key residues involved in this network as 
 well as the corresponding hydrogen - bonding distances in the 3srg crystal structure 
 are shown . despite great progress in this 
 area , our knowledge of how interfacial activation of enzymes by protein 
lipid 
 interactions regulates enzyme stability , efficiency , and specificity 
 still remains limited .
as pon1 shares many structural features with 
 other interfacially activated enzymes , including in particular secreted 
 phospholipase 2 ( spla2 ) , it provides a promising model 
 system with which to understand the molecular details of such interfacial 
 activation and , following from this , also a further probe for the 
 role of enzyme dynamics in catalysis .
structural considerations have 
 identified a key hydrogen - bonding network that spans from the protein 
membrane 
 interface to the catalytic calcium center , interacting in particular 
 with n168 , which interacts with the substrate and plays a role in 
 fixing the position of the catalytic calcium , as well as y71 , which 
 lies on the active site loop and interacts with residues in the central 
 tunnel ( figure 1b ) .
the importance of this hydrogen - bonding network 
 can be in part highlighted by the fact that disturbing even its peripheries 
 by removal of hdl or detergents or mutation of key second- or third - shell 
 residues such as d183 causes a complete loss in catalytic activity .
our recent combined biochemical , crystallographic , 
 and computational study suggested that interaction with hdl rigidifies 
 this hydrogen - bonding network , keeping the key catalytic residues 
 in place and thus leading to the observed stimulation of pon1 s 
 lactonase activity .
this also touches 
 on the  dark side  of excess enzyme dynamics and the 
 need to sometimes reduce floppiness to enhance activity ( a phenomenon 
 also seen in directed evolution of several de novo - designed enzymes ) .
a key remaining question is what the role of the active 
 site loop , 
 and in particular y71 , is in facilitating pon1 s activity , 
 specificity , and promiscuity and how it compares to other organophosphatases 
 with similar active site loops .
our recent computational work , which 
 probed the effect of distorting the h2 helix on the catalytic activity , 
 showed only minor displacement of y71 in response to the repositioning 
 of h2 when pon1 was in complex with the chromogenic lactone thiobutyl--butyric 
 lactone ( tbbl ) .
taken together , this 
 would suggest that y71 does not play an active role in pon1 s 
 lactonase activity in general , and specifically not on the hdl - induced 
 simulation .
however , as can be seen from figure 1b , y71 is a key participant in the hydrogen - bonding 
 network connecting the catalytic calcium center to the membrane interface 
 of pon1 .
additionally , when the disordered loop is closed over the 
 active site , y71 points directly into the active site , which makes 
 it much harder to fit larger and bulkier organophosphate substrates 
 such as the pesticide paraoxon into the active site , even though kinetic 
 characterization shows that pon1 is able to hydrolyze paraoxon quite 
 efficiently , with kcat / km = 2149  393 m s. both docking and molecular 
 dynamics ( md ) simulations suggest that 
 the only way paraoxon can be accommodated in the pon1 active site 
 is by pushing y71 out of the active site , causing the active site 
 loop to take on a partially open conformation .
this raises key questions 
 about the role of y71 and , following from this , about the role of 
 active site plasticity in facilitating a catalytically favorable active 
 site architecture in this highly promiscuous interfacially activated 
 enzyme .
while the most direct way to probe the importance of 
 the conformation 
 of y71 and the active site loop on catalysis would be through crystal 
 structures of pon1 in both its substrate - free form and in complex 
 with different inhibitors , enzyme  ligand complexes of pon1 
 are notoriously difficult to crystallize , and 
 therefore , we have explored the role of y71 and active site plasticity 
 indirectly through a combination of site - saturation mutagenesis of 
 y71 ( using a small library that contains the variation of y71 to all 
 other 19 amino acids ) and screening for both lactonase activity toward 
 tbbl and paraoxonase activity .
doing so has allowed 
 us to explore how different residues at this position in the active 
 site loop would affect the configuration of pon1 s binding 
 site and in turn both its native and promiscuous activities . our data 
 show that while the effect of mutation of y71 on the observed catalytic 
 activity is not radical , as expected , it affects both the loop flexibility 
 and the rigidity of the hydrogen - bonding network connecting the active 
 site to the surface helices .
the observed changes in active site architecture 
 upon substitution of y71 can also to some extent help rationalize 
 the kinetic observations from previous studies with other pon1 variants .
following from this , the 
 resulting structural reshaping of the 
 active site also affects substrate positioning , which can be directly 
 linked to the observed changes in catalytic activity .
more significantly , 
 however , the y71d183 hydrogen - bonding interaction 
 appears to allow the active site loop to act as a hydrophobic gate , 
 allowing movement of the substrate and product in and out of the active 
 site and , more critically , helping to exclude solvent from the active 
 site , thus providing a hydrophobic cage in which the reaction can 
 take place .
mutations of y71 cause changes in the active site volume 
 that allow an increasing number of solvent molecules access to the 
 active site , although this is more pronounced when the organophosphate 
 is bound to the active site compared with tbbl , which can in turn 
 be directly linked to the observed differential effects of the same 
 mutation on the lactonase and paraoxonase activities .
this influx 
 of water molecules will modulate the electrostatic environment of 
 the active site , thus also impacting the corresponding catalytic efficiency 
 of the enzyme .
a comparison to other organophosphatases demonstrates 
 that these enzymes often either have an active site loop that appears 
 to play a similar role or have found other solutions to create a hydrophobic 
 cage for the substrate ( e.g. , through a deeply buried active site 
 pocket ) .
we posit that this regulation of the electrostatic environment 
 of the active site through solvent exclusion , which is clearly advantageous 
 for the binding of hydrophobic molecules such as lactones and aryl 
 esters , is key to the fortuitous ability of these enzymes to also 
 hydrolyze uncharged organophosphate nerve agents and pesticides .
this 
 creates a major difference with other promiscuous phosphatases such 
 as members of the alkaline phosphatase superfamily , where active site polarity and charge discrimination 
 between different anionic substrates apparently play key roles in 
 facilitating the observed selectivity and promiscuity .
the site - saturation library in position 71 
 was generated using the 
  inverse polymerase chain reaction  method with the g2e6 variant of pon1 as the template , 
 as previously described .
the g2e6 variant 
 is a chimeric form of pon1 ( by directed evolution ) showing functional 
 and structural properties similar to those of human pon1 .
however , unlike the latter , the g2e6 variant 
 is highly soluble and capable of bacterial expression , which thus 
 enabled detailed analyses of pon1 s structure and function . for simplicity , in the text
we refer to the g2e6 wild - type - like 
 variant as repon1 throughout .
the tyrosine codons 
 in position 71 were replaced with nns codons ( where n is a mixture 
 of all four nucleotides and s is a mixture of c and g ) encoding all 
 amino acids .
the library was transformed into origami cells , and colonies 
 were grown on agar plates .
randomly picked colonies were individually 
 grown in 96-deep - well plates , and protein production was induced with 
 iptg as previously described .
following cell lysis , 
 clarified lysates of the y71 variants were screened for phosphotriesterase 
 ( paraoxon ; 1 mm ) and lactonase ( tbbl ; 0.2 mm ) activities .
mutants 
 with interesting activity phenotypes were subjected to dna sequencing 
 and further characterization .
the expression 
 and purification of repon1 and its y71 mutants were performed as previously 
 described .
kinetic measurements with tbbl 
 and paraoxon were determined at substrate concentrations in a range 
 from 0.3  km up to ( 23 ) 
  km , and kcat , km , and kcat / km were obtained by fitting 
 the data to the michaelis  menten model with prism ( graphpad 
 software ) .
all of the data presented are 
 the means of at least two independent experiments , and the error ranges 
 represent the standard deviations from the mean .
the starting 
 point for all of the pon1 simulations in this work was a 2.2  
 crystal structure of repon1 in complex with the inhibitor 2-hydroxyquinoline 
 ( 2hq ) and obtained at ph 6.5 ( pdb i d 3srg ) .
we chose 
 this structure in particular as it is one of the only crystal structures 
 of pon1 in the protein data bank in which the active site loop is 
 sufficiently stabilized by the inhibitor to be observed in the crystal 
 structure .
for comparison , we have simulated both the substrate - free 
 enzyme and the enzyme  substrate complexes of repon1tbbl 
 and repon1paraoxon in both the wild - type ( wt ) and mutant forms 
 of the enzyme .
because of the lack of crystal structures , the relevant 
 y71 mutant forms of repon1 were generated in silico either by manual 
 truncation of y71 to phe , ala , or gly or by choosing the most probable 
 rotamer of met and trp using the dunbrack rotamer library as implemented in chimera . in all cases , and as in our previous work , the relevant substrate 
 and the nucleophilic water molecule were manually placed into the 
 active site so as to optimize the alignment of the nucleophilic water 
 molecule and the reactive c / p center , the alignment of the nucleophilic 
 water molecule and the relevant general base , and the alignment of 
 the substrate with amino acid side chains in the active site . in both 
 cases ,
the conformation of 2hq in the 3srg crystal structure was used as a guiding 
 point for where to place the substrate into the active site .
additionally , 
 both the structural and catalytic calcium ions were modeled using 
 an octahedral dummy model overlaid on the position 
 of the relevant metal ion in the crystal structure and the same parameters 
 as in our previous study ( see table s1 ) .
this had the advantage of allowing 
 for more stable metal coordination and avoided the local structural 
 instabilities we observed when using a standard lennard - jones sphere 
 as a calcium model , which were also independently alluded to in ref ( 54 ) . to solvate the 
 system , all of the crystallographic water molecules were removed from 
 the crystal structure , except for those directly coordinated to the 
 ca ions , and the system was solvated in a 24  - radius 
 sphere of tip3p water molecules centered on the catalytic ca ion .
this was then subjected to a 10 kcal mol  surface restraint on all atoms in the 
 outer 15% of the sphere to keep it stable , as per the surface - constrained 
 all - atom solvent model ( scaas ) .
all atoms 
 outside this sphere were retained in the simulation but restrained 
 to their crystallographic positions using a 200 kcal mol  harmonic positional restraint , and all 
 atoms within the inner 85% of the sphere were allowed to move freely 
 ( also see our previous work ) . in order 
 to keep the charge of the sphere stable , all ionizable groups within 
 17  of the catalytic ca were kept in their relevant 
 protonation states at physiological ph , and all other ionizable groups 
 outside and close to the boundary of the sphere were kept in their 
 neutral forms , resulting in an overall system charge of 1 
 ( table s2 ) . once the system had been solvated 
 and correct ionization states had been assigned , each structure was 
 gradually heated from 0 to 300 k over the course of 240 ps of simulation 
 time , starting with a 200 kcal mol  positional restraint on all heavy atoms and reducing 
 this restraint to 0.5 kcal mol  on all atoms of the substrate and nucleophilic water molecule ( when 
 present in the simulation ) over the course of the heating procedure . 

once the system had reached 300 k , we performed a further 30 ns of 
 md simulations to monitor the behavior of the loop and equilibrate 
 the system for subsequent empirical valence bond ( evb ) simulations 
 of the chemical step of catalysis .
all simulations were performed 
 in three replicas with different initial velocities using the q simulation 
 package and the opls - aa force field with a 1 fs time step and the berendsen thermostat 
 with a 1 fs bath relaxation time , leading to a total of 1.62 s 
 of ( productive ) simulation time over all systems .
we note as 
 an aside here that , as outlined in the introduction , pon1 is a membrane - associated enzyme , and extensive 
 delipidation has been shown to lead to complete loss of activity . as in our previous study
, we did not 
 explicitly include the membrane in our simulations both because of 
 the excessive computational cost this creates when running large numbers 
 of simulations of the enzyme - catalyzed hydrolysis of multiple substrates 
 by both wild - type and mutant forms of repon1 and because of the lack 
 of any direct structural information on how pon1 , or in fact any other 
 enzyme , interacts with hdl . however , we demonstrated in our previous 
 study that even without explicitly including the membrane in our simulations , 
 they still provided meaningful information because we are working 
 with a multilayer system in which the outer peripheries of repon1 
 were kept restrained to their crystallographic coordinates .
this restraint 
 approach , which is standard practice in qm / mm simulations of enzyme 
 reactivity ( see , e.g. , refs ( 60 ) and ( 61 ) ) , mimics to some extent the stabilizing effect of the membrane or 
 detergent and prevents the protein from artificially unfolding in 
 the absence of the membrane while keeping the active site structure 
 stable .
following from this , we demonstrated in our previous study 
 that our model allowed us to reproduce both relevant activation free 
 energies and other key experimental observables with high fidelity , 
 increasing our confidence in the reliability of our simulations ( see 
 the discussion in ref ( 25 ) ) , and once again , in the present work we were able to reproduce 
 the relevant energy changes upon mutation of y71 using our model ( see 
 the results and discussion ) .
following from our initial molecular 
 dynamics simulations of wild - type and mutant forms of repon1 , we have 
 modeled the effect of mutating y71 on the energetics of tbbl and paraoxon 
 hydrolysis using the evb approach , which is an empirically based hybrid 
 vb / mm approach that uses classical force fields to model chemical 
 reactivity within a quantum - mechanical framework .
the full theoretical details of this approach have been 
 reviewed at great length elsewhere , but in brief , the two 
 main advantages of this approach in the context of the present work 
 are ( 1 ) its lower computational cost as a classical approach , which 
 allowed us to perform the extensive conformational sampling necessary 
 to obtain convergent free energies for chemical processes in biological 
 systems , and ( 2 ) the fact that a well - parametrized force field carries 
 a tremendous amount of chemical information , allowing us to model 
 bond breaking and formation processes in a chemically meaningful way 
 ( in the evb approach , this is achieved using morse rather than harmonic 
 potentials to describe bonds that are changing ) . on the basis 
 of previous experimental and computational studies , here we describe the hydrolyses of paraoxon and tbbl as one- and 
 two - step reactions , respectively ( figure 2 ) , using the valence bond states illustrated 
 in figure s1 . in our recent evb study ,
we considered only the nucleophilic attack step 
 to form a tetrahedral intermediate for tbbl hydrolysis , as this step 
 is most likely to be rate - limiting .
however , in the present study , 
 for completeness we have also considered the subsequent ring - opening 
 step .
nucleophile activation has been modeled as proceeding through 
 general base catalysis by the side chains of h115 and d269 for tbbl 
 and paraoxon hydrolysis , respectively , on the basis of extensive mutagenesis 
 data ( also see our previous 
 work ) , and in the case of tbbl hydrolysis , 
 we modeled the ring - opening step with proton transfer back from the 
 protonated h115 side chain , which now acts as a general acid catalyst 
 instead , as this is the most likely proton source .
proposed mechanisms of 
 ( a ) paraoxon and ( b ) tbbl hydrolysis by 
 serum paraoxonase 1 .
all of the evb simulations for each system were performed 
 by taking 
 the end point of one of our replicas from the initial md simulations 
 for each system and preparing 10 new starting structures by running 
 another 500 ps of md simulations with a new set of initial velocities 
 for each starting structure .
the subsequent evb simulations were then 
 performed from these new starting structures using the standard evb 
 free energy perturbation umbrella sampling ( fep / us ) approach outlined 
 in , e.g. , refs ( 6264 ) , and all of the evb 
 simulations for each system were performed in 51 mapping frames of 
 200 ps / frame , for simulation times of 10.2 ns for each individual 
 system for paraoxon hydrolysis and 20.4 ns for tbbl hydrolysis , as 
 the latter was modeled as a two - step process ( 306 and 612 ns per system , 
 respectively , over 30 independent evb trajectories , considering three 
 independent initial md simulations as starting points and then 10 
 evb simulations initiated from each individual equilibrated system ) . 

this leads to a total simulation time of 5.51 s over all systems 
 ( 5.69 s when including also the initial 500 ps md simulations ) . 

the evb simulations were performed using the same setup as our initial 
 md simulations and applying a weak 0.5 kcal mol  harmonic constraint on all free reacting 
 fragments during the simulation to prevent them from drifting out 
 of the simulation sphere . in each case , our reference state was the 
 uncatalyzed reaction in aqueous solution , the evb parameters for which 
 were calibrated to fit activation and reaction free energies of g1 = 20.7 kcal mol , gint = 14.4 kcal mol , and g2 = 17.5 kcal / mol for tbbl hydrolysis ( where g1 and 
 g2 are the free energies of activation 
 for nucleophilic attack and ring opening , respectively , and gint is the free energy of the corresponding 
 tetrahedral intermediate ) and g = 23.9 kcal mol and g0 = 10.5 kcal mol for paraoxon 
 hydrolysis .
this calibration was based on extensive consideration 
 of existing experimental and computational data and is described in detail in the supporting information of ref ( 25 ) .
the evb simulations of 
 the background reaction in aqueous solution were performed using the 
 same setup as for the wild - type enzyme , with ethylimidazole and propionic 
 acid as models for h115 and d269 , respectively .
the only differences 
 in simulation setup between the reactions in water and the enzyme 
 active site were that we used only 10 ns of equilibration time for 
 the water reaction and that a slightly larger harmonic restraint of 
 1 kcal mol  ( compared 
 with 0.5 kcal mol  for 
 the corresponding enzyme - catalyzed reactions ) was placed on the reacting 
 atoms to keep them at the center of the simulation sphere .
once each 
 background reaction had been calibrated to reproduce the relevant 
 energetics in aqueous solution , the same parameter set was then used unchanged in order to model the reactivities of the wild - type 
 and mutant forms of repon1 with each substrate .
the resulting calculated 
 activation free energies were obtained using the qcalc module of q , 
 and unless otherwise specified , all other external analysis was performed 
 using the cpptraj module as implemented 
 in amber 16 . all of the evb parameters 
 necessary to reproduce our work
an examination of the structure of repon1 ( figure 1 ) strongly suggests 
 that the active site loop of repon1 ( defined here as residues 7081 , 
 following ref ( 7 ) ) 
 and related enzymes acts as a lid , facilitating both entrance of the 
 substrate to the active site and subsequent product release .
however , in addition to the overall 
 structural role of this loop , previous work has shown that y71 in 
 particular is an important residue that plays a key role in pon1 s 
 function .
this includes , for example , modulation of substrate selectivity 
 and catalytic stimulation by hdl .
the side chain of y71 is highly mobile and has been 
 shown to adopt various conformational substates that have been suggested to differentially affect pon1 s 
 native and promiscuous functions .
therefore , 
 in order to further probe the role of residue y71 , we have generated 
 a saturation library at position 71 ( i.e. , substitution of y71 with 
 all 20 amino acids ) and used it for library screening of both the 
 chromogenic lactone tbbl and the organophosphate pesticide paraoxon . 
 in general , substitutions at position 71 mainly affected the paraoxonase 
 activity .
only 16% of the tested library mutants ( see materials and methods ) exhibited paraoxonase activity 50% 
 compared with y71 , whereas the lactonase activity was retained in 
 52% of the library mutants .
the mutants showing the most diverse functional 
 properties in the library screens ( i.e. , y71 m , y71w , y71f , y71a , and 
 y71 g ) , such as differential activity between repon1 s native 
 ( lactonase ) and promiscuous ( paraoxonase ) activities , were purified , 
 and we experimentally measured the rates of hydrolysis of both tbbl 
 and paraoxon by each repon1 variant ( table 1 ) .
these mutations potentially introduce 
 significant structural perturbations to the active site of pon1 , which 
 consequently affect its function .
thus , further analysis of these 
 mutants may allow us to examine both the structural impact of y71 
 mutations on the active site configuration , especially with regard 
 to the flexible loop , and their corresponding effect on the activity 
 and selectivity of repon1 toward its native ( lactonase ) and promiscuous 
 ( paraoxonase ) substrates .
kinetic parameters 
 were measured 
 at 25 c and ph 8 , and kcat , km , and kcat / km are shown in s , mm , and 
 m s , respectively . because of limited substrate solubility , 
 the reaction rates did not show saturation ; thus , kcat could not be determined , and only an upper limit for 
 the km value based on the maximal substrate 
 concentration used is provided . the catalytic efficiency was obtained 
 by linear fit of the initial velocity to the substrate concentration . 

gexp denotes the experimental activation 
 free energy obtained as an upper limit from kcat using transition state theory , and gcalc denotes the corresponding calculated activation free energy obtained 
 from our empirical valence bond ( evb ) simulations using the evb parameters 
 shown in the supporting information .
the 
 gcalc values shown in this table are averages 
 and standard deviations over 30 independent trajectories as described 
 in materials and methods .
all of the activation 
 energies are shown in kcal mol . for a detailed 
 description of the derivation of the energetics of the background 
 reaction in aqueous solution and the corresponding evb calibration 
 used to obtain the calculated values shown in this table
, we refer 
 the reader to materials and methods and the 
 supporting information of ref ( 25 ) . in the case 
 of tbbl hydrolysis , 
 the calculated activation free energies shown here correspond to the 
 rate - limiting step for a two - step process ( figure 2 ) . for a breakdown of the calculated energies 
 for each individual reaction step ,
representative structures of the corresponding stationary points 
 for each reaction step for the reactions catalyzed by the wild - type 
 enzyme are shown in figures s5 and s6 . as can be seen from figure 1 , in the wild - type 
 enzyme y71 forms a hydrogen - bonding interaction 
 with the side chain of d183 , which is itself part of a hydrogen - bonding 
 network that leads from n168 on the catalytic ca ion 
 to the outer periphery of the protein .
d183 is central to this hydrogen - bonding 
 network , as in addition to the hydrogen bond it forms with the side 
 chain of y71 , it also forms hydrogen - bonding interactions with the 
 side chains of s166 , n168 , and h184 , the backbone carbonyl group of 
 s166 , and an active site water molecule .
therefore , d183 acts as an 
  anchor  , keeping this hydrogen - bonding network together . 

the importance of the hydrogen - bonding network in general and residue 
 d183 in particular is further supported by the fact that mutations 
 at position 183 have been shown to be detrimental to both pon1
s 
 lactonase and paraoxonase activities , even though this residue is 7  away from the 
 catalytic ca and makes no obvious contacts with the bound 
 substrate in the active site .
our starting point , therefore , 
 was to examine the structural implications 
 of the y71 substitutions and to see the extent to which breaking the 
 hydrogen bond between y71 and d183 affects the dynamics of the active 
 site loop and the hydrogen - bonding network along the central tunnel . 

the most direct way to examine this would be to generate crystal structures 
 of the relevant mutant forms of the enzyme ; however , the active site 
 loop , including y71 , is disordered and absent from almost all of the 
 few existing pon1 structures in the protein data bank .
recently , we 
 were able to make significant progress in this direction by using 
 an inhibitor , 2hq , to obtain a structure of repon1 at ph 6.5 with 
 a closed loop conformation and a visible side chain at position 71 .
however , as was previously shown , mutations in the flexible loop , especially at positions 
 71 and 74 ( both make direct contact with 2hq ) , abolish the binding 
 of 2hq . therefore , crystallization of the y71 mutants , even in the 
 presence of 2hq , is likely to produce crystal structures with a disordered 
 active site loop , including the side chain at position 71 . because 
 of this , we are dependent on simulation in order to elucidate the 
 effect of mutations on the positioning and dynamics of the active 
 site loop . therefore , we began by performing md simulations 
 of repon1 and 
 each of the y71 mutants mentioned above in either the substrate - free 
 form of the enzyme or in complex with either paraoxon or tbbl ( three 
 30 ns trajectories for each system ) , as described in materials and methods . the root - mean - square deviations ( rmsds ) 
 of all backbone c atoms during our md simulations 
 ( shown in figure s2 and table s3 ) demonstrate 
 the stability of our system during our simulations and that the system 
 adequately equilibrated during the time scale of the simulations . 

similarly , a comparison of the metal  ligand distances between 
 the catalytic ca ion and its surrounding ligands ( table s4 ) shows that the metal center remained 
 stable during our simulations , and that while the metal 
ligand 
 distances were slightly perturbed by the y71 mutations , they were 
 highly stable throughout our simulations without the need for any 
 restraints or artificial bonds to the ligands .
we have also 
 examined the root - mean - square fluctuations ( rmsfs ) 
 of the backbone c atoms during the course of our 
 md simulations ( figure s3 ) and observed 
 that in all of the simulations , the regions of the protein with the 
 largest fluctuations are the active site loop ( residues 7081 ) , 
 a second loop ( residues 4655 ) that is close to the metal - bound 
 e53 , and a shorter loop segment ( residues 133140 ) that is 
 adjacent to h134 , which forms part of the h115h134 catalytic 
 dyad in the active site .
as would be expected , 
 the simulations of repon1 in complex with either tbbl ( figure s3b ) or paraoxon ( figure s3c ) show the flexibility of the active site loop either 
 to be similar to that in the wild - type enzyme or to slightly increase 
 upon substitution of y71 ( as a result of breaking of the y71d183 
 hydrogen bond ) .
interestingly , however , binding of paraoxon to the 
 active site appears to greatly reduce the flexibility of the active 
 site loop , even though it increases the active site volume compared 
 with those of the corresponding repon1tbbl complexes , as shall 
 be discussed below . additionally , comparing the wild - type and mutant 
 forms of repon1 in both the substrate - free forms and in complex with 
 paraoxon and tbbl ( figure s4 ) illustrates 
 that despite these changes in flexibility , the active site loop is 
 still very floppy and can take multiple conformations .
therefore , 
 the loop itself appears to be moving back and forth in the simulations , 
 and there is not a direct correlation between either loop position 
 and mutation or loop position and which substrate is bound to the 
 active site , except that substrate binding appears to significantly 
 increase the overall mobility of the enzyme compared with the substrate - free 
 form ( figure s3 ) . as mentioned in the previous section , 
 the y71 substitution library overall seems to affect more drastically 
 the promiscuous paraoxonase activity compared with the native lactonase 
 activity ( 16% vs 52% of screened variants showing 50% paraoxonase 
 and lactonase activity , respectively , compared with the y71 wt ) .
the 
 kinetic parameters determined for the individual y71 mutants also 
 largely agree with this observation ( table 1 ) .
that is , it can be seen , for instance , 
 that kcat / km is reduced by up to 25-fold for tbbl hydrolysis and by up to 120-fold 
 for paraoxon hydrolysis , with the y71 m and y71 g mutants showing the 
 largest decrease , respectively . for most of the mutants , however , km is minimally affected by the mutation , and 
 thus , the changes in activity primarily arise from changes in kcat .
an exception is the y71w mutant , which 
 shows higher km values for both substrates , 
 presumably due to structural hindrances induced by the bulkier side 
 chain of trp , which might affect substrate binding .
more curiously , 
 the same mutation apparently impacts the activities of repon1 toward 
 the two substrates differently .
that is , in the case of tbbl hydrolysis , 
 the most detrimental effect on kcat is 
 observed in the conservative mutation of tyr to phe ( which involves 
 only loss of a hydrogen bond ) , whereas much smaller effects are observed 
 upon replacement of y71 with ala or even gly , although none of the 
 observed effects are particularly large for any of these mutations 
 in free energy terms .
in contrast , in the case of paraoxon hydrolysis , 
 the trend is reversed , with only a small effect from mutation to met , 
 a slightly larger effect from mutation to phe , and then a significant 
 effect of truncation to either gly or ala , resulting in a 100-fold 
 reduction in kcat ( corresponding to a 
 2.8 kcal / mol change in activation free energy ) .
overall , our 
 experimental results clearly show a differential effect of the y71 
 mutations on repon1 s native and promiscuous functions .
specifically , 
 the y71 mutations seem to have a much larger effect on paraoxon hydrolysis 
 ( promiscuous function ) than on tbbl hydrolysis ( native function ) . 

this observation is interesting , as the shape of the active site wall 
 is complementary to the native substrate , even though y71 has no direct 
 contacts with the lactone ( it should be noted , however , that binding 
 of the promiscuous organophosphate substrate is driven by nonspecific 
 hydrophobicity ) .
this is therefore in agreement 
 with the suggestion that native functions are more robust and resistant 
 to mutations compared with promiscuous functions ( although recent work suggests that this may not 
 be valid for all systems ) .
that is , it 
 has been argued that mutations that have a neutral or near - neutral 
 effect on the native function might remarkably affect promiscuous 
 function(s ) and have thus been suggested to serve as a driving force 
 for the evolvability of promiscuous functions .
similarly to y71 , these mutations are often located in 
 the flexible regions of the binding - site walls , such as loops .
thus , 
 in the present case , it is plausible that variations in the composition 
 of the flexible loop , especially at position 71 , have contributed 
 greatly to the evolvability of pon1 s paraoxonase activity . to further probe the origin of these differential changes in pon1 s 
 native and promiscuous activities
, we have performed evb simulations 
 of the repon1-catalyzed hydrolysis of tbbl and paraoxon as described 
 in materials and methods , and our data are 
 summarized in figure 3 and table 1 . on the 
 basis of extensive experimental studies of pon1-catalyzed tbbl hydrolysis , we have modeled tbbl hydrolysis as proceeding through general base 
 catalysis by h115 and paraoxon hydrolysis as proceeding through general 
 base catalysis by the metal - coordinated d269 , in line with our previous 
 work .
we note that the geometries of 
 nucleophilic attack are different for tbbl and paraoxon hydrolysis , 
 with the scissile bond of paraoxon needing to be in - line with the 
 attacking water molecule , whereas in 
 the case of tbbl the nucleophile attacks from a brgi 
dunitz 
 angle of 102. however , the fact 
 that pon1 appears to use different general bases for nucleophile activation 
 for the two substrates resolves this issue , as the substrates occupy 
 different positions in pon1 s binding pocket , placing them 
 in the correct position relative to the nucleophile in each case ( because 
 of steric constraints , paraoxon can not bind in a conformation that 
 would allow for in - line attack by a water molecule that is deprotonated 
 by h115 ) ( figure 1b ) . 

this causes the two substrates to use different subsets of active 
 site residues to catalyze their respective hydrolysis reactions , which 
 has been suggested to be one of the reasons for pon1 s selectivity 
 and promiscuity .
in addition , even though the 
 overall substrate positions are different , in these two cases the 
 c = o and p = o ester bonds overlay almost perfectly ( figure 1b ) , allowing for 
 activation of the ester bond by the catalytic calcium ion ( also see 
 the discussion in refs ( 10 ) and ( 25 ) ) . comparison 
 of calculated ( green ) and experimental ( blue ) activation 
 free energies for the hydrolysis of ( a ) tbbl and ( b ) paraoxon by wild - type 
 repon1 and the y71 variants of interest in this work . all energies 
 are in kcal mol and correspond to the values shown 
 in table 1 .
error bars 
 correspond to the standard deviations of over 30 individual evb trajectories , 
 which were generated as described in materials and 
 methods . in the case of tbbl hydrolysis , the calculated activation 

free energies correspond to the rate - limiting step for a two - step 
 process ( figure 2 ) . 
 for a breakdown of the calculated energies for each individual reaction 
 step ,
representative structures 
 of the corresponding stationary points for each reaction step for 
 the reactions catalyzed by the wild - type enzyme are shown in figures s5 and s6 . using this model , we are able to reproduce the effect of 
 mutations 
 of y71 on the energetics of paraoxon hydrolysis with high accuracy 
 and to obtain excellent quantitative agreement with the experimental 
 data ( figure 3 and table 1 ) . in addition
, we 
 can clearly reproduce the increase in activation free energy observed 
 for paraoxon hydrolysis upon perturbation of y71 , with a steady increase 
 in the calculated activation free energy that is in line with the 
 experimental observations . in the case of tbbl hydrolysis , our 
 calculated activation free 
 energies appear to be slightly too low compared with the corresponding 
 experimental values , and in addition , the y71w substitution appears 
 to disrupt the stability of the active site , considering the large 
 standard deviation we obtain in the calculated activation free energy . 
 following from this
the lower activation free energies could be due 
 to the calibration of the corresponding reference reaction in aqueous 
 solution , to which we fit our evb parabola , as this value is not known 
 experimentally for tbbl hydrolysis and we were therefore forced to 
 fit to quantum - chemical calculations of hydroxide attack on analogous 
 lactone substrates .
if our parametrization 
 of the energetics of imidazole - catalyzed tbbl hydrolysis in aqueous 
 solution underestimates the activation barrier for this process , this 
 will cause the corresponding enzyme - catalyzed reactions to have artificially 
 low activation free energies .
in addition , correlating the calculated 
 experimental activation free energies for pon1-catalyzed tbbl hydrolysis 
 is more complex than for paraoxon hydrolysis for two reasons : ( 1 ) 
 the actual experimental energy differences in the case of tbbl hydrolysis 
 are extremely small ( up to only a 1.2 kcal mol increase relative to the wild type across the series of mutants ; 
 see table 1 ) and thus 
 very hard to reproduce computationally , and ( 2 ) the 
 measured kcat actually 
 corresponds to breakdown of the final product to release a thiol moiety , 
 which is detected by using ellman s reagent ( see ref ( 4 ) ) , and thus only provides 
 us with an upper limit for the activation free energy for the lactone 
 hydrolysis by each variant . however , our calculated values are never 
 larger than the experimental values , and also , they never vary by 
 more than about 2 kcal mol across the series of 
 all six repon1 variants studied here , which , when the standard deviations 
 of the calculated values are also taken into account , shows much less 
 sensitivity to the y71 substitutes than is observed in the case of 
 paraoxon hydrolysis .
this increases our confidence in the ability 
 of our model to rationalize the origin of these differential effects 
 of the y71 substitutions on the two substrates . from our md 
 simulations of the michaelis complex
, it was clear 
 that although the loop dynamics is affected by mutation of y71 , there 
 is no direct trend between the behavior of the loop and the specific 
 mutation ( i.e. , the loop flops back and forth ) , and thus , the position 
 of the active site loop alone is not the origin for the changes in 
 paraoxonase activity upon mutation of y71 . however , there are significant 
 differences in the position of the side chain of residue 71 upon mutation , 
 with concomitant effects on the overall organization of the active 
 site residues ( figures s4 and s7 ) .
the 
 most conservative substitution is that of y71 into phe , where the 
 hydrogen - bonding interaction with d183 is lost because of the absence 
 of the oh group .
the model structures from our simulations 
 reveal significant changes in the orientation of the f71 side chain 
 compared with that of the wild - type y71 . for instance , in the absence 
 of substrate , the f71 side chain is oriented into a hydrophobic region 
 located below the flexible loop and formed by residues l69 , i74 , and 
 v346 .
this in turn perturbs the configuration of this hydrophobic 
 region in order to accommodate the bulkiness of the phe side chain 
 ( figure s7 ) . in the presence of either 
 paraoxon or tbbl ,
however , the f71 side chain is pushed toward outside 
 the active site , which results in the opening of the flexible loop 
 as well as changes in the configuration of h134 .
the substitution 
 of y71 into trp leads to very similar observations ( figure s7 ) .
interestingly , the residues forming the 
 hydrophobic region ( i.e. , 
 l69 , i74 , and v346 ; figure s7 ) were shown 
 to play a critical role in pon1 s function and specificity , 
 especially toward paraoxonase activity .
for instance , it was shown 
 that truncation of i74 into ala reduced the paraoxonase activity by 
 > 25-fold , while the lactonase activity was not affected .
moreover , mutations of both l69 and v346 were 
 shown by several studies to increase the organophosphatase activity 
 of pon1 ( e.g. , mutations l69s
this could be due to changes in the configuration 
 of the hydrophobic region upon mutation of these residues , which would 
 affect the accommodation of bulky organophosphate side chains such 
 as the o - ethyl side chains of paraoxon .
substitution 
 of y71 with either ala or gly also seems to induce 
 structural changes not only to the loop but also to the residues of 
 the hydrophobic region .
the absence of the bulkiness of y71 seems 
 to affect specifically the orientations of residues l69 , i74 , and 
 v346 ( figure s7 ) .
in contrast , as a result 
 of the flexible nature of the y71 m side chain , it occupies a position 
 very similar to that of y71 , with minimal perturbations to the active 
 site .
this side chain is also relatively bulky and flexible and can 
 accommodate multiple configurations , and therefore , one would not 
 expect a significant perturbation upon substitution with met .
overall , 
 this analysis suggests that mutation at position 71 might also induce 
 an indirect effect on the active site configuration , especially by 
 changes in the orientations of residues l69 , i74 , and v346a , which 
 in turn would be expected to have a functional impact on pon1 activity .
clearly these structural perturbations reshape the active site , 
 thus impacting the catalytic activity of the enzyme .
p / o  c distances between 
 the nucleophile and substrate as well as the distances associated 
 with deprotonation of the nucleophilic water molecule , where perturbations 
 appear to primarily affect the position of the nucleophile relative 
 to the substrate and relative to the general base ( tables s6 and s7 ) .
we note additionally that we can not directly 
 compare these calculations to previous qm cluster and qm / mm studies of paraoxon hydrolysis , 
 as these have primarily focused on the alkaline hydrolysis of paraoxon 
 catalyzed by enzymes with bimetallo active sites . however , despite 
 this limitation , we have obtained similar concerted and highly associative 
 transition states as in refs ( 87 ) and ( 88 ) , which is in good agreement with the experimental data on both enzymatic 
 and nonenzymatic paraoxon hydrolysis and quantum - chemical 
 calculations on the uncatalyzed reaction .
following from this , in an examination of the average occupancies 
 ( % ) of hydrogen bonds in the hydrogen - bonding network along the active 
 site tunnel at both the michaelis complex and the transition state 
 for each substrate ( tables s8 and s9 , respectively ) , 
 it can be seen that , in particular , mutation of y71 disrupts hydrogen - bonding 
 interactions between n168 and the substrate as well as interactions 
 involving s166 and d183 as well as s166 and k192 .
the disruption of 
 the hydrogen bond to k192 is particularly relevant , as we demonstrated 
 in our recent study that the correct 
 positioning of this residue is crucial to the paraoxonase activity 
 of pon1 .
the importance of a positive charge at this position can 
 also be seen from the effects of pon1 192r / q polymorphs upon interaction 
 with hdl .
that is , in human pon1 both in vitro and in vivo , the 192q 
 isozyme exhibits significantly lower hdl binding and lipolactonase 
 activity than the 192r polymorph . in 
 addition ,
recombinant pon1 - 192k and pon1 - 192r show similar stability 
 and lipolactonase activity upon association with hdl , whereas recombinant 
 pon1 - 192q shows both reduced stability and lipolactonase stimulation .
finally , in our computational work , we demonstrated that the effect of k192 on the 
 paraoxonase activity is much larger than that on the lactonase activity , 
 which may explain part of the observed differential effect of the 
 y71 mutations , which disrupt the hydrogen - bonding network keeping 
 this residue in place , on the two activities .
however , while it is 
 clear that mutation of y71 both affects the positioning of the active 
 site loop and reshapes critical interactions in the active site pocket 
 and along the central tunnel , these structural changes do not follow 
 a specific trend with different mutations or explain why paraoxon 
 hydrolysis is so much more affected by mutation of y71 than tbbl hydrolysis . 
 what , then , is the role of y71 and the active site loop and where 
 do these differences come from ? up to this point
, we 
 have not taken into account changes in penetration of water into the 
 active site upon y71 modifications that lead to increased opening 
 of the active site loop .
this can be significant , as both paraoxon 
 and tbbl are hydrophobic substrates , and , as can be seen from figure 4 , once the active 
 site lid closes over the substrate , this creates a hydrophobic cage 
 with only the nucleophilic water molecule in the immediate vicinity 
 of the reacting atoms .
this then alters the local electrostatic environment 
 of the active site , which would be expected to strengthen the effect 
 of local electrostatic interactions through lowering of the local 
 dielectric constant . as expected , and as shown in figure 4 for the example of the y71 g 
 variant , substitutions of y71 also affect the degree of solvent accessibility 
 in pon1 s active site . to examine the extent to which this 
 is affected by mutation of y71 and breaking of the y71d183 
 hydrogen bond , we have calculated the average numbers of water molecules 
 within 6  of the reacting parts of tbbl and paraoxon , defined 
 as the carbonyl group and ring oxygen of tbbl and the phosphorus atom 
 and connecting oxygen atoms of paraoxon , during our md simulations 
 of the michaelis complex ( table 2 ) .
we note that the numbers of active site water molecules 
 shown in table 2 for 
 simplicity do not include the nucleophilic water 
 molecule and refer only to water molecules coordinating to the catalytic 
 calcium ion and the mobile nonreactive water molecules in the active 
 site .
the corresponding values at the transition state and for the 
 solvation of the nitro moiety of paraoxon are shown in tables s10 and s11 , respectively .
these tables 
 demonstrate that the michaelis complex trends are largely retained 
 in the transition state and that the main changes are observed in 
 the proximity of the reacting atoms rather than the nitro group .
comparison 
 of solvent accessibility in representative active sites 
 of repon1 in complex with ( a , b ) paraoxon and ( c , d ) tbbl in the michaelis 
 complexes of the wild - type and y71 g repon1 variants , respectively . 
 the solvent - accessible area is shown in shaded gray , and all water 
 molecules that are within 6  of the reacting centers are shown 
 explicitly .
the number of water 
 molecules in 
 the active site refers to the number of water molecules within 6  
 of the reacting atoms of the substrate ( defined as the carbonyl group 
 and ring oxygen of tbbl and the phosphorus center and connecting atoms 
 of paraoxon ) , calculated as averages over the last 25 ns of three 
 molecular dynamics trajectories using visual molecular dynamics .
these numbers are not integers because they 
 are averages over all snapshots from our molecular dynamics simulations . 

the active site volume was calculated 
 with pocket volume measurer , version 2.0.1 , using the default settings based on a sphere with an 8  radius 
 located at the pocket center ( 7 ,  21 ,  31 ) . 
 as can be seen from the data , substitution of y71 not only increases 
 the average active site volume but also the standard deviation of 
 the active site volume , suggesting an increase in active site flexibility .
solvent denotes the contribution of the interaction between the reacting 
 atoms and solvent molecules to the total calculated reorganization 
 energy ,  , in kcal mol .
the data shown here 
 are averages and standard deviations of the pocket volume over the 
 last 25 ns of our equilibration runs for each system , averaged over 
 three independent trajectories ( i.e. , a total of 75 ns of simulation 
 time over the three trajectories ) . as can be seen from table 2 , and as expected , the truncation of y71 
 to a smaller side 
 chain affects the active site similarly for both paraoxon and tbbl . 

however , while in both cases we observe an increase in the number 
 of water molecules , for paraoxon the relative effect across the series 
 of mutations seems slightly more significant .
that is , while there 
 are originally more water molecules near the reacting atoms when tbbl 
 is bound to the active site , the number of water molecules within 
 6  of the tbbl ring during our simulations increases by only 
 two additional water molecules moving closer to the substrate upon 
 truncation of y71 to either ala or gly compared with the wild - type 
 enzyme .
in contrast , in the case of paraoxon , where the bulky ethyl 
 groups of the substrate push on the active site loop , the mutation of y71 allows a steadily increasing number 
 of water molecules to access to the active site , going from only one 
 water molecule in the wild - type enzyme to six water 
 molecules on average for the y71 g variant ( also see figure 4 ) .
this difference is interesting 
 in light of the much smaller effect of mutations of y71 on the lactonase 
 activity than on the paraoxonase activity . in parallel , from an examination 
 of the changes in active site volume upon substitution of y71 ( table 2 ) , it seems that there 
 is a clear increase in active site volume upon substitution of y71 , 
 which appears to follow the decrease in the paraoxonase activity of 
 repon1 .
we note that table 2 shows not only the average volumes over the course of our 
 md simulations but also the corresponding standard deviations in these 
 values .
this gives an estimate of how  floppy  the active 
 site is during the simulations ( the larger the standard deviation , 
 the more the active site volume changes ) .
these data show not only 
 that the active site volume increases upon substitution of y71 but 
 also that the active site becomes floppier .
finally , in order to quantify 
 the contribution of the increasing number of water molecules to the 
 calculated activation free energies , we have also applied the linear 
 response approximation to our calculated evb trajectories in order 
 to extract solute 
solvent , the contribution 
 of solute  solvent interactions ( where the  solute  
 is defined as the reacting atoms ) to the total reorganization energy 
  ( table 2 ) . 

this provides a quantitative measure of the system reorganization 
 upon moving between the different evb states ( in kcal mol ) , where a larger reorganization energy corresponds to a higher the 
 activation free energy and vice versa ( see clarifications in , e.g. , 
 refs ( 63 ) and ( 93 ) ) . as can be seen from 
 these data , the change in the solute 
solvent reorganization 
 energy across the series of repon1 variants is much larger in the 
 case of paraoxon hydrolysis than in the case of tbbl hydrolysis , by 
 a factor of 2 . taken together , these data suggest that the 
 role of y71 is to act as a hydrophobic gate , keeping the active site 
 loop closed and thus restricting solvent access to the active site , 
 in turn creating a hydrophobic cage for the hydrolysis of hydrophobic 
 lactones and neutral organophosphates . in view of the fact that 
 many enzymes capable of organophosphatase 
 activity are primarily
either lactonases or have evolved from lactonases , this raises the intriguing question of whether active site hydrophobicity 
 and mobile active site loops play a similar role in other organophosphatases . 

the most immediate point of comparison is the enzyme dfpase , which 
 hydrolyzes diisopropylfluorophosphate ( dfp ) and has an active site 
 virtually identical to that of pon1 ( figure 5 ) .
the key differences between the two active 
 sites are the absence of a histidine at a position structurally equivalent 
 to h115 in pon1 , which presumably leads to the observed lack of lactonase 
 activity in this enzyme ( although other 
 enzymes that lack a histidine at this position have shown lactonase 
 activity ) , and the fact that the dfpase loop , which is the closest 
 corresponding loop to pon1 s active site loop , is far more 
 rigid and does not act as a lid over the active site . while both pon1 and dfpase share a similar six - bladed -propeller 
 fold with two ca ions in the central tunnel , pon1 has 
 a unique addition of three helices covering the binding site that 
 form the hdl - binding interface , which 
 is not present in dfpase . more significantly , however , a more recently 
 characterized lactonase with relatively high organophosphatase hydrolase 
 activity from the phosphotriesterase - like lactonase family , ssopox , has an active site loop that undergoes major conformational 
 changes upon lactone binding and thus provides a hydrophobic channel 
 to the active site .
another active site loop has 
 been implicated in regulating the promiscuous organophosphatase activity 
 of a lactonase from geobacillus kaustophilus hta426 . finally , several other organophosphatases , 
 such as the bacterial phosphotriesterase ( pte ) , acetylcholine esterase , and 
 organophosphate hydrolase , have largely 
 hydrophobic active sites , and such active site hydrophobicity has 
 been suggested to play a role in the multispecificity of these enzymes . in the case of pte , it has also been possible 
 to introduce lactonase activity through selective active site loop 
 modeling , where shortening a key active site loop minimally reduces 
 the paraoxonase activity ( 45-fold ) while allowing the emergence 
 of homoserine lactonase activity .
it 
 is important to note that these enzymes not only come from very different 
 organisms but also have both very different overall secondary structures 
 and active site architectures , and thus , creating a hydrophobic binding 
 pocket ( whether through active site gates like pon1 s active 
 site loop or through having deeply buried active sites ) is the only 
 significant structural feature these enzymes have in common .
comparison 
 of the tertiary structures of ( a ) repon1 ( pdb i d 3srg ) and ( b ) dfpase 
 ( pdb i d 1e1a ) , with the active site loops highlighted in orange .
( c ) overlay 
 of the catalytic ca and coordinating residues for the 
 two enzymes , with repon1 shown in blue and dfpase shown in green .
interestingly , we recently performed 
 a similar comparison of the 
 structures and physicochemical properties of highly promiscuous members 
 of the alkaline phosphatase superfamily , showing a correlation between the active site volume , the polar 
 solvent - accessible surface area , and the number of characterized promiscuous 
 activities in the literature . in the case of the alkaline phosphatases , 

these enzymes preferentially hydrolyze a range of mono- and dianionic 
 phosphate and sulfate esters , and where paraoxonase activity has been measured for members of 
 this superfamily , it has been extremely low . in contrast , for example , the bacterial phosphotriesterase , which 
 has evolved to hydrolyze hydrophobic and neutral lactones and organophosphates , 
 is a very poor catalyst of charged phosphodiesters .
therefore , the active site architectures that these enzymes 
 have evolved to catalyze reactions involving charged substrates appear 
 to be quite different from those evolved to catalyze neutral and hydrophobic 
 organophosphates such as paraoxon and dfp .
active site architecture 
 and charge discrimination are therefore clearly very important in 
 determining the specificity and promiscuity in both sets of reactions , 
 as not only do these bulky hydrophobic substrates prefer the solvent - excluded 
 environment created by , for instance , closing of the active site loop 
 of pon1 , but also , in a low - dielectric environment , electrostatic 
 interactions in the active site will be magnified , leading to much greater stabilization of , for instance , 
 the oxyanion intermediate created during lactone and ester hydrolysis . 

therefore , these enzymes have presumably evolved the ability to form 
 hydrophobic cages in order to facilitate their native lactonase / esterase 
 activities ( for which the substrates are generally more hydrophobic ) , 
 and the corresponding organophosphatase activities coincidentally 
 happen to have similar chemical requirements as the native lactones / aryl 
 esters , taking advantage of the architecture the enzyme provides for 
 facilitating the native reaction .
we note also that the importance 
 of hydrophobic residues in facilitating organophosphate hydrolysis 
 has also been discussed in other studies of individual organophosphatases 
 ( e.g. , refs ( 77 ) , ( 96 ) , and ( 99 ) ) and in the case of acetylcholine 
 esterases , where the hydrophobic binding site shows sensitivity to both the hydrophobicity of inhibitors and to mutations of hydrophobic active site 
 residues .
the flexible loop in the mammalian pon family ( i.e. , residues 7081 
 in pon1 s numbering ) consists of both conserved and variable 
 positions .
the most conserved positions are 72 and 73 ( pro and gly , 
 respectively ) , which seem to have a structural role in shaping the 
 flexible loop as a  lid  that covers the binding site . 
 on the other hand , positions 74 and 75 are the most diverse and show 
 different preferences for each family member ( e.g. , pon1-i74 , pon2-l74 , 
 and pon3-m74 ) .
position 71 also shows different preferences , such 
 that while both pon1 and pon3 have tyr and show paraoxonase activity , 
 pon2 has either phe or cys ( rarely ) and shows no detectable paraoxonase 
 activity .
similarly , the bacterial pon ( ponx - occal ) , 
 which has arg in position 71 , also shows no detectable paraoxonase 
 activity .
interestingly , the predicted 
 mammalian ancestor of the pon family , n9 , which shows a  generalist  activity profile , including 
 paraoxonase activity , has a tyr in position 71 . it is therefore appealing 
 to speculate that y71 enables an optimized binding - site configuration 
 for the binding and catalysis of organophosphates .
indeed , in attempts 
 at directed evolution of pon1 toward efficient catalysis of organophosphates 
 ( including nerve agents ) , y71 was substituted with various alternative 
 side chains , but tyr was retained in all variants with improved catalytic 
 activities . in the present work , 
 we have probed the role of the tyr at position 
 71 further by performing detailed experimental and computational analyses 
 of the role of selected y71 variants in the active site stability 
 and catalytic activity of pon1 .
we have demonstrated that while the 
 measured kcat for the lactonase activity 
 is perturbed only up to 8-fold upon substitution of y71 , the corresponding 
 paraoxonase activity is perturbed 100-fold .
clearly , substitution 
 of y71 changes the active site by creating volume at this position , 
 and y71 plays a role in positioning all of the other participants 
 in the hydrogen - bonding network along the central tunnel in the appropriate 
 positions for substrate binding and catalysis .
when this residue is 
 removed , our simulations show that the new residue at position 71 
 is displaced from the original y71 position , either through side - chain 
 rotations in the case of the trp / phe substitutions or through being 
 pushed out of the active site by the spectator ethyl groups of paraoxon . 

therefore , substitutions at this position have a much larger effect 
 on the active site architecture than would be anticipated , which in 
 turn affects both substrate binding and the catalytic activity of 
 the enzyme . from a structural perspective , studying the effect of 
 substitutions at position 71 also highlights the potential roles of 
 residue d183 in catalysis .
that is , this residue , which forms a hydrogen 
 bond to y71 ( figure 1 ) , has been shown to be completely immutable , despite the 
 fact that it makes no direct contacts with either the lactone or organophosphate 
 substrates .
previous work highlighted the significance of this residue 
 but did not rationalize why this position is sensitive to substitution . 

our computational analysis demonstrates that d183 appears to play 
 an important role in  catching 
y71 to close the active 
 site lid , rather than the other way around , as the loop does not fly 
 out when this interaction is removed .
in addition , we have demonstrated 
 that while substitution of y71 
 clearly has an impact on the flexibility of the active site loop , 
 the largest effect of substituting y71 appears to be disrupting the 
 position of k192 ( which is a very important residue for modulating 
 pon1 activity ) as well as , most critically , 
 affecting the active site hydrophobicity .
that is , substitution of 
 y71 expands the active site volume and allows for an increasing number 
 of water molecules to enter the active site , an effect that is unsurprisingly 
 most pronounced upon substitution of y71 with either ala or gly .
this 
 effect is far more pronounced when paraoxon is present in the active 
 site than when tbbl is present in the active site and thus appears 
 to be the most likely origin of the differential effect of the y71 
 substitutions on the two activities .
this differential effect is also 
 interesting in light of studies showing that enzymes native 
 activities are far more robust toward mutations than their promiscuous 
 activities , and such minor changes in the 
 native activity and large changes in the promiscuous activity hint 
 at the greater evolvability of promiscuous functions .
a comparison 
 of the active site architecture of pon1 to those of 
 other organophosphatases demonstrates that either a similar  gating  
 loop or a deeply buried active site that excludes solvent is a common 
 feature of these enzymes .
in addition , as with related promiscuous 
 phosphatases such as members of the alkaline phosphatase superfamily , 
 charge discrimination still plays a major role in determining the 
 selectivity .
that is , in the case of the alkaline phosphatase superfamily , 
 these enzymes are promiscuous catalysts that preferentially facilitate 
 the hydrolysis of mono- and dianionic substrates and show only poor 
 activity toward neutral organophosphate esters such as paraoxon .
in contrast , in the case of 
 organophosphate hydrolases , these enzymes preferentially catalyze 
 large hydrophobic substrates such as lipolactones or neutral phosphate 
 esters , and where for example the diesterase activity has been measured , 
 it has been significantly lower than the triesterase activity despite 
 the similar compact and associative transition states expected for 
 the hydrolysis of these two phosphate esters .
we therefore posit that this ability to generate a hydrophobic active 
 site cage to enclose the substrate is a key feature in facilitating 
 the evolution of organophosphatase activity in these promiscuous enzymes , 
 which are often primarily ( in turn hydrophobic ) lipolactonases or 
 arylesterases .
in addition to being of fundamental interest from a 
 functional evolution perspective , these insights can also be utilized 
 in order to rationally design next - generation catalysts .
that is , 
 a number of studies have explored the role of water in both enzyme 
 engineering studies and engineered binding sites 
 for drug design .
we have demonstrated 
 here as well that selective hydration of the active site allows pon1 
 to discriminate between its native lactonase and promiscuous organophosphatase 
 activities .
therefore , if one can , for example , open up a space to 
 allow more water molecules into the active site , this might be a rational 
 way to limit one enzymatic activity without increasing or impacting 
 the other .
therefore , active site hydration can also be used as a 
 tool to control the artificial evolution of enzyme function , allowing 
 the design of enzymes to select the substrate of a specific reaction 
 from a pool of viable substrates .
 serum 
 paraoxonase 1 ( pon1 ) is a native lactonase capable of promiscuously 
 hydrolyzing a broad range of substrates , including organophosphates , 
 esters , and carbonates . 
 structurally , pon1 is a six - bladed -propeller 
 with a flexible loop ( residues 7081 ) covering the active site . 
 
 this loop contains a functionally critical tyr at position 71 . 
 we 
 have performed detailed experimental and computational analyses of 
 the role of selected y71 variants in the active site stability and 
 catalytic activity in order to probe the role of y71 in pon1 s 
 lactonase and organophosphatase activities . 
 we demonstrate that the 
 impact of y71 substitutions on pon1 s lactonase activity is 
 minimal , whereas the kcat for the paraoxonase 
 activity is negatively perturbed by up to 100-fold , suggesting greater 
 mutational robustness of the native activity . 
 additionally , while 
 these substitutions modulate pon1 s active site shape , volume , 
 and loop flexibility , their largest effect is in altering the solvent 
 accessibility of the active site by expanding the active site volume , 
 allowing additional water molecules to enter . 
 this effect is markedly 
 more pronounced in the organophosphatase activity than the lactonase 
 activity . 
 finally , a detailed comparison of pon1 to other organophosphatases 
 demonstrates that either a similar  gating loop  or 
 a highly buried solvent - excluding active site is a common feature 
 of these enzymes . 
 we therefore posit that modulating the active site 
 hydrophobicity is a key element in facilitating the evolution of organophosphatase 
 activity . 
 this provides a concrete feature that can be utilized in 
 the rational design of next - generation organophosphate hydrolases 
 that are capable of selecting a specific reaction from a pool of viable 
 substrates . 
Introduction
Materials and Methods
Results and Discussion
Overview and Conclusions