american trypanosomiasis , 
 also known as chagas disease , 
 is caused by the parasite trypanosoma cruzi ( t. cruzi ) and is considered by the world health organization 
 ( who ) to be one of the 17 neglected tropical diseases .
it is endemic 
 in central and south america where it affects 1012 million 
 people , killing over 15,000 each year and infecting hundreds of thousands 
 worldwide .
it is transmitted to humans through the 
 feces of triatomine bugs known as  kissing bugs  , and 
 also between humans by blood transfusion and from mother to infant .
the acute phase of the disease is usually asymptomatic 
 while the chronic phase is characterized by the development of cardiac 
 and digestive pathologies .
while most trypanosomatid 
 parasites remain in blood , t. cruzi is an intracellular 
 parasite .
this fact seriously hinders the development of drugs for 
 the treatment of chagas disease . to date ,
nifurtimox and benznidazole , are the only two approved 
 drugs for the treatment of the infection despite their low efficacy 
 and their severe side effects .
consequently , 
 investigations of new and more effective drugs as well as the characterization 
 of novel targets are required .
the trans - sialidase 
 from t. cruzi ( tcts ) is essential for the parasite 
 infectivity in the human body , and combined with the fact that tcts 
 is not present in humans , it is currently considered a promising biochemical 
 target for developing new molecules to control chagas disease .
sialic acids are o- and n - substituted derivatives of neuraminic 
 acid , a nine - carbon monosaccharide , usually located on cell surface 
 glycoproteins and glycolipids .
these acids play a key role in biology 
 by regulating antigenic expression and molecular interactions and 
 providing structural support and protection to cell membranes .
sialidases are a family of enzymes that catalyzes the removal of 
 sialic acid from various glycoconjugates and constitute virulence 
 factors of numerous viruses and prokaryotic and eukaryotic microorganisms . these enzymes are expressed in different parasites 
 including trypanosoma brucei , the causative agent 
 of african trypanosomiasis , trypanosoma rangeli ( t. rangeli ) , and t. cruzi .
both the sialidase of t. 
 rangeli ( trsa ) and tcts are part of the sialidase family , 
 but while the former is a strict hydrolase , the latter preferentially 
 displays a trans - sialidase activity catalyzing the 
 transfer of sialic acid residues from the host glycoconjugates to 
 the parasite surface mucins . as a result
, the parasite is protected from the host s immune 
 system and gains the ability to adhere to and invade host cells .
tcts and trsa have a 70% sequence identity 
 and the overall c root mean squared deviation ( rmsd ) 
 value is only 0.59 
  .
the n - terminal 
 catalytic domain shows a -propeller fold which is connected 
 by a long -helical segment to the c - terminal domain displaying 
 a -barrel lectin - like topology . despite the 
 high structural similarity ,
no trans - sialidase activity 
 is present in trsa , which exclusively catalyzes the hydrolysis of 
 sialic acid residues from sialyl - glycoconjugates .
furthermore , good inhibitors of trsa ( e.g. , dana and tamiflu ) are 
 only very weak inhibitors of tcts , indicating that small differences in the sequence 
 and structure can give rise to substantial changes in the catalytic 
 mechanism . in this work
, the residue numbers of both trsa and 
 tcts will be 
 referred to those of the crystal structure of the tcts complex with 
 sialyllactose ( pdb i d : 1s0i ) .
the active sites of 
 tcts and trsa contain several residues conserved in other microbial 
 sialidases .
those active site residues include the following : the 
 catalytic residues tyr342 , glu230 , and asp59 ( involved in proton shuffling 
 and nucleophilic attack ) ; an arginine triad ( arg35 , arg245 , and arg314 ) , 
 which interacts with the carboxylate group of the sialic acid ; and 
 asp96 , which is h - bonded with o11 and n12 of sialic acid . in addition , 
 in tcts , trp312 and tyr119 display a hydrophobic lactose - binding region . 
 in trsa , tyr119 is substituted by ser119 leading to a less suitable 
 region for the accommodation of the lactose ring .
the catalytic 
 mechanisms of the hydrolysis and transfer reaction 
 are depicted in scheme 1 .
once the cilac is 
 obtained , only tcts is able to transfer sialic acid to the parasite 
 lactose - containing mucins leading to the mclac .
if the 
 lactose group in the cilac is replaced by a water molecule 
 at the active site of the enzyme , the newly established ciwat is hydrolyzed by a water molecule to mcwat .
trsa is a 
 very efficient hydrolase while tcts preferentially shows trans - sialidase activity .
structural and kinetic 
 studies revealed a double - displacement ( ping - pong ) 
 mechanism with formation of a covalent sialyl - enzyme intermediate 
 via tyr342 .
after the formation of the michaelis complex in the presence of 
 lactose ( mclac ) , both enzymes are able to cleave the host 
 sugar  sialic acid bond leading to the covalent intermediate , 
 with the leaving group ( donor sugar ) still present at the active site 
 ( cilac ) .
once the cilac is reached , tcts completes 
 its trans - sialidase activity by taking the reverse 
 step but in this case transferring the sialic acid to a parasite sugar 
 ( acceptor group ) leading to a new mclac . in this reaction , 

the anomeric carbon of the sialic acid is attacked by the lactose 
 hydroxyl oxygen , which is deprotonated by asp59 while the tyr342 phenolic 
 oxygen is protonated by glu230 .
in contrast , trsa is unable to achieve 
 the transfer reaction of sialic acid to the parasite surface glycoconjugates . 
 in this case
, the donor sugar in cilac is replaced by a 
 water molecule ( ciwat ) .
although this second path can be accomplished by both enzymes , tcts 
 was shown to be significantly more efficient in transferring than 
 in hydrolyzing sialic acids .
a 
 great deal of effort has been applied to identify the determinants 
 of the trans - sialidase activity in tcts in contrast 
 with the strict hydrolase activity of trsa .
the active site of tcts 
 shows a narrower and more hydrophobic binding pocket than trsa .
this 
 was confirmed by molecular dynamics ( md ) simulations performed by 
 our group that showed that the trsa ciwat adopted a more 
 open catalytic cleft compared to that of the tcts , facilitating the 
 access of water molecules .
therefore , 
 the trans - sialidase activity in tcts can , in part , 
 be favored by the exclusion of water molecules and the establishment 
 of nonpolar interactions between the active site residues and the 
 lactose group .
moreover , we have recently found 
 that when lactose is not present in the active site , the catalytic 
 couple tyr342-glu230 is dissociated when tcts is found in its covalent 
 intermediate ( ci ) form .
these facts support the idea of 
 a long - lived ci that could be involved in the sialyl - transfer mechanism 
 of tcts .
( met95-val , ala97-pro , 
 ser119-tyr , gly248-tyr and gln283-pro ) leading to a quintuple mutant 
 that showed around 1% trans - sialidase activity .
however , despite the numerous experimental and 
 theoretical studies carried out thus far , the reasons for the different 
 catalytic activities between both enzymes are still unclear .
molecular modeling has become one of the most important tools to 
 gain atomic insight and quantitative understanding of enzymatic reactions .
computational enzymology provides detailed features 
 of the reaction pathways acting as a complement to experimental techniques .
recently , we studied the cilac formation in tcts and in trsa , together with specific mutations 
 that provided important insight into the trans - sialidase 
 activity . in the present work ,
the hydrolytic 
 activity of tcts and trsa was modeled using computer simulations and 
 free energy calculations in order to complete the mechanistic studies 
 in tcts and trsa and rationalize their distinct catalytic behavior .
md simulations 
 of the ciwat of trsa and tcts ( pdb ids : 2a75 and 2ah2 , respectively ) were 
 performed by our group and described in previous work .
representative conformations of tcts and trsa 
 from unrestrained md simulations were used as initial structures for 
 the quantum mechanics / molecular mechanics ( qm / mm ) umbrella sampling 
 simulations .
those systems included a water molecule present at a 
 location suitable for the nucleophilic attack . in this conformation
, 
 a hydrogen bond is established between the water molecule and the 
 carboxylic group of asp59 .
the distance between the oxygen atom of 
 the water molecule and the anomeric c in both covalent intermediates 
 is less than 4  and an average hydroxyl oxygen ( tyr342)anomeric 
 c ( sialic acid)o ( water ) angle of approximately 170 
 suggested an ideal position for an in - line attack on the anomeric 
 c ( sialic acid)hydroxyl oxygen ( tyr342 ) bond .
classical molecular 
 mechanics can provide atomic insight into the active site of enzymes 
 and contribute to identifying the determinants of reactivity . however , 
 they are unable to treat processes taking place during enzymatic reactivity , 
 including bond formation and breaking . in the hybrid
qm / mm approach , the reactive part is treated using 
 quantum mechanics while the rest of the system is treated with a classical 
 force field ( mm ) . for each ciwat ,
the qm region included glu230 , asp59 , sialic acid , tyr342 , arg245 , 
 arg314 , and arg35 and the water molecule while the rest of the enzyme 
 residues were treated using the amber99sb force field ( figure 1 ) .
representative structure of the tcts covalent 
 intermediate ( ciwat ) used as the starting point for the 
 umbrella sampling simulations . 

the amino acids included in the classical mm region are shown in green 
 while the qm residues are displayed in light blue sticks .
hydrogen link atoms were used to treat the covalent bonds 
 between 
 the side chain of the catalytic residues ( qm region ) and the backbone 
 of the protein ( mm region ) .
the self - consistent charge density functional 
 tight binding ( scc - dftb ) method , as implemented in amber , was employed to study the quantum region .
scc - dftb 
 was used successfully in many biomolecular simulations and chemical 
 reactions and was found to be in good agreement with qm / mm 
 calculations developed at higher levels of qm theory , such as mp2 .
this method was also shown to provide the best 
 semiempirical description of five- and six - membered carbohydrate ring 
 deformations .
we have previously shown 
 that the energy barriers obtained using dftb and mp2 for the transsialidase activity of tcts are very similar.- the reactions of the hydrolysis of the 
 ciwat to mcwat were modeled by means of umbrella 
 sampling using conditions 
 similar to those previously used in tcts .
the distances involved in defining the reaction coordinates ( denoted 
 as rc1 and rc2 ) are depicted in scheme 2 .
the 2-d reaction coordinate 
 is defined by rc1 and rc2 : rc1 = d3  d4 and rc2 = d1  d2 .
d1 refers to the distance between the 
 anomeric carbon and the catalytic water oxygen while d2 refers to the distance between a asp59 carboxylic oxygen 
 and the nearest water hydrogen .
d3 refers 
 to the distance between the anomeric carbon and the tyr342 hydroxyl 
 oxygen , and d4 corresponds to the distance 
 between one glu230 carboxylic oxygen and the tyr342 hydroxyl oxygen . 

a combination of two reaction coordinates was used to model the reaction : 
 rc1 represents the cleavage of the sialic acid  tyr342 bond 
 and the protonation of the tyrosine by abstracting a proton from the 
 glu230 oxygen : rc1 = d3  d4 .
rc2 describes the deprotonation of the water 
 molecule by an asp59 oxygen and the subsequent attack of the oxygen 
 to the anomeric carbon of the sialic acid : rc2 = d1  d2 .
rc1 was harmonically 
 restrained from 0.30 to 2.10  while rc2 was restrained 
 from 5.9 to 2.4  .
the scans were completed using 
 0.08  steps with a force constant of 450.0 kcal/(mola ) .
the total number of windows in the 2-d umbrella calculation 
 was around 1300 . for each
qm / mm simulation 2 ps was considered to 
 equilibrate the system before running 20 ps in the production phase . 

for every md simulation , periodic boundary conditions with a cutoff 
 distance of 12.0  and a time step of 1.0 fs were used .
the particle 
 mesh ewald method was used to calculate the long - range coulomb forces . the 2-d weighted histogram analysis method ( wham-2d )
was used to unbias the simulations and to obtain 
 the free energy surface ( fes ) along the reaction coordinates following 
 the same protocol used for the study of the trans - sialidase activity of tcts .
the minimum 
 free energy path ( mfep ) is identified by following the path connecting 
 the energy minima through the transition state ( ts ) that is orthogonal 
 to the equipotential contours of the fes .
the energy decomposition 
 method was employed to investigate the functional roles of tcts and 
 trsa active site residues during the hydrolysis of the sialic acid 
 ciwat .
this method has been widely applied to identify 
 the importance of different amino acids in the catalytic mechanism 
 of enzymes and was recently employed by our group in different studies .
we used it here to investigate the stabilization pattern of tcts 
 and trsa residues on tswat with respect to the ciwat .
the qm region included the catalytic residues asp59 , glu230 , and 
 tyr342 , a water molecule , and the sialic acid .
a total of 400 configurations 
 in the ciwat and tswat configurations were selected 
 in each case .
for each configuration , every residue from the mm subsystem 
 within 10  to the sialic acid was individually mutated to gly . 

the averaged quantum mechanical energy was computed in each step in 
 the wild - type proteins and for each mutant structure .
the influence 
 of a particular residue on the energy of a particular conformation 
 was measured taking into account the difference of energies when a 
 particular residue is present ( denoted by i in eq 1 ) or when it is replaced by gly ( i  1 in eq 1).1 the contribution of each residue to 
 the stabilization of tswat during the hydrolysis of the 
 ciwat to the mcwat was obtained by measuring 
 the difference between the stabilization patterns of these residues 
 in tswat and ciwat,2thus , a positive / negative 
 value of ei indicates that residue i exerts a higher destabilization / stabilization 
 electrostatic 
 effect on tswat than on ciwat .
this selective 
 stabilization pattern occurs because the analyzed residue adjusts 
 its position and/or because the electrostatic distribution of the 
 qm subsystem changes when the system goes from ciwat to 
 tswat .
thus , the e values 
 represent useful information on how the enzyme selectively stabilizes 
 a particular state ( i.e. , tswat ) with respect to another . 

it should be noted that the relationship between the e and how a mutation can help to further stabilize the reaction 
 is not direct .
in fact , if we modified a specific residue , not only 
 the electronic distribution of the qm subsystem , but also the positioning 
 of the other residues are affected , and thus all of the pattern is 
 modified . in order 
 to describe the mechanism , a reaction space plot based on a two - dimensional 
 more oferral  jencks diagram was created for both trsa and tcts enzymatic reactions .
the reactant 
 ( ciwat ) is located on the lower left corner , the product 
 is on the upper right , and the x and y axes represent the pauling bond order along the mfep for o ( water)sialic acid bond making and 
 the breaking of the sialyl 
the pauling 
 bond order , nx , was determined 
 using the following equation : where n0 denotes 
 the bond order of the fully formed bond of length r0 ( in this case r0 = 1.4  , 
 and n0 = 1 ) and rx is the average distance between the leaving 
 group ( tyr342 ) or nucleophile ( water ) and the anomeric c. three - dimensional 
 structures and trajectories were visually inspected by using the computer 
 graphics program pymol .
interatomic distances 
 and angles were monitored by using the ptraj module 
 in ambertools 12 .
the analysis 
 of the 2-d fes describing the hydrolysis of the ciwat by 
 tcts and trsa showed a clear mfep connecting ciwat to mcwat ( figure 2 ) .
free energy surfaces 
 for the hydrolysis of the ciwat catalyzed by tcts ( left ) 
 and trsa ( right ) .
the white dashed lines illustrate the minimum energy path 
 to reach mcwat from ciwat . for both enzymes mfeps
proceed diagonally indicating that 
 the reaction 
 coordinates rc1 ( involving tyr342sialic acid bond cleavage ) 
 and rc2 ( associated with the water activation and nucleophilic attack 
 on the anomeric c ) are highly correlated . to better describe the mechanism , 
 a  reaction space 
plot , based on a more oferral  jencks 
 style diagram , is presented in figure 3 .
reaction space for the hydrolysis of the ciwat during 
 the sialidase activity of tcts ( red ) and trsa ( black ) . for both systems , at the transition state ( tswat ) , the 
 tyr342sialic acid bond is almost cleaved ( pauling bond order 
  0.1 ) while the csial  owat bond 
 formation is beginning ( pauling bond order  0.26 ) , showing 
 that the hydrolysis reaction follows an andn mechanism of dissociative type .
interestingly , the same behavior was observed 
 for the trans - sialidase activity of both enzymes .
taking into account all of these data , it can be concluded that , 
 for tcts and trsa , the mechanism is andn dissociative , regardless of the nature 
 of the nucleophile or the leaving group . in others words ,
the sialidase 
 and trans - sialidase activities share a common mechanism 
 in both cases .
the last paragraph highlights common features 
 of the hydrolysis 
 mechanism of tcts and trsa
. however , it is worth noticing some subtle 
 aspects that differ , such as the conformations of the reaction critical 
 points , depicted in figure 4 . structures obtained from 
 the qm / mm umbrella sampling simulations 
 corresponding to the covalent intermediate ( ciwat ) , transition 
 state ( tswat ) , and final michaelis complex ( mcwat ) , for the hydrolysis of sialic acid catalyzed by tcts ( top ) and 
 trsa ( bottom ) . the initial ciwat displays a very similar structure 
 between tcts and trsa .
the water molecule is hydrogen - bonded to asp59 
 and is suitably positioned for a nucleophilic attack to the sialic 
 acid anomeric carbon . at the tswat , tyr342
is no longer 
 bound to sialic acid and its hydroxyl oxygen is protonated by glu230 . 

a hydrogen bond is still present between the water molecule and asp59 , 
 but the covalent bond with the sialic acid is not yet formed .
the distance from the oxygen atom of the 
 nucleophilic water molecule to the anomeric c of sialic acid is the 
 same for trsa and tcts ( d1 = 2.1  ) . 

in contrast , d2 is 0.4  shorter 
 in trsa than in tcts , making the oxygen atom of the water molecule 
 a better nucleophile in the former . at the tswat of both 
 enzymes ,
the tyr342sialic acid bond is already cleaved and 
 tyr342 has accepted the proton from glu230 .
this proton transfer is 
 almost completely achieved in both tcts and trsa ( d4 = 1.3 and 1.2  , respectively ) .
significant 
 conformational changes of the sialic acid ring were 
 observed during the hydrolysis of the ciwat to mcwat . in the cilac ,
the structures sampled at 
 the tswat adopted a h5 half - chair 
 conformation where atoms o5 , c1 , c4 , and c5 form the reference plane . finally , when the mcwat is reached , 
 the sialic acid ring conformation migrates to a b2,5 boat
. 
 we can follow this process by computing the pyramidalization dihedral 
 around the anomeric carbon of sialic acid ( figure 5 ) .
 o13 distance ( red ) and c9 ( anomeric c)c10o13c19 
 dihedral angle ( green ) along the minimum free energy path in tcts 
 ( top ) and trsa ( bottom ) .
the torsion moved from + 40 
 ( ciwat ) to 40 
 ( mcwat ) describing a reverse conformation change compared 
 to the one observed during the trans - sialylation 
 reaction in tcts , resulting in an overall retention of the configuration 
 of the sialic acid ring .
the evolution of the 
 anomeric c  o13 bond distance along the mfep was also analyzed . 

the distance is shortened gaining a partial double bond character 
 at the tswat , after which the anomeric c  o13 bond 
 recovers the single bond character reproducing the effects observed 
 during the transfer activity in tcts .
the energy barrier 
 ( gr ) to reach 
 the mcwat from the ciwat in tcts is 26.8 kcal / mol , 
 which is considerably higher than the barrier obtained for trsa ( 16.4 
 kcal / mol ) , indicating that the hydrolysis of ciwat is clearly 
 more favored in trsa .
these data are in good agreement with the experimental 
 evidence that identifies trsa as a more efficient hydrolase than tcts .
although trsa and tcts share very similar structural 
 components , the active site shows important differences that could 
 account for the different hydrolysis rates of both enzymes .
moreover , 
 the modifications observed in tcts are able to overcome the fact that 
 the water concentration in the active site is several orders of magnitude 
 higher than that of lactose such that hydrolysis should be theoretically 
 favored over the transfer reaction .
the calculated energetic values of the hydrolysis catalyzed by tcts 
 can be compared with those from the trans - sialidase 
 activity obtained in our previous works so that we can complete the 
 rationale of the different activity of both enzymes ( figure 6 ) .
free energy profiles for the trans - sialidase 
 ( top ) 
 and sialidase ( bottom ) activities catalyzed by tcts and trsa . in tcts , the calculated energy 
 barrier to bind a second lactose 
 to the covalent intermediate during the trans - sialidase 
 reaction is 20.8 kcal / mol6 kcal / mol lower than the hydrolysis 
 reaction .
this agrees with the experimental findings confirming that 
 tcts behaves preferentially as a trans - sialidase 
 rather than as a sialidase .
furthermore , the free energy difference 
 ( gr ) between the product 
 of the hydrolysis ( mcwat ) and reactant ( ciwat ) in tcts is 9.7 kcal / mol , indicating that the ciwat is 
 much more stabilized than the mcwat . on the other hand 
 gr between the product of trans - sialylation ( mclac ) and the reactants ( cilac )
is 0.9 kcal / mol making the cilac approximately 
 equienergetic with the mclac .. these values suggest that although tcts can behave as a sialidase 
 at a much lower rate , the formation and stabilization of a ci is strongly 
 preferred over its hydrolysis to the mcwat .
these findings 
 attest to the formation of a long - lived ci in tcts as it is required 
 during the trans - sialidase activity by retaining 
 the sialic acid longer , which favors the binding of new lactose to 
 sialic acid .
the hydrolysis and trans - sialylation 
 energy barriers 
 were also compared for trsa .
we noticed that the hydrolysis reaction 
 barrier is roughly 10 kcal / mol lower than that of the transfer reaction . 
 as a consequence
, the hydrolysis in trsa will be strongly preferred 
 over the trans - sialylation .
in addition , gr between mcwat and ciwat is 3.7 kcal / mol , whereas gr between mclac and cilac is 10.9 
 kcal mol , making mcwat a much more 
 stable complex than mclac in trsa .
these results suggest 
 that the reactions are kinetically controlled and explain why trsa 
 does not show any trans - sialidase activivity and 
 behaves as a strict hydrolase .
moreover , as it was shown for tcts , 
 the formation of a ci in trsa is favored due the lower energy barriers 
 calculated to reach the ci from the mc . in trsa
the trans - sialidase activity energy values of 
 tcts were also compared to the hydrolysis activity measurements in 
 trsa . according to experimental data , the hydrolysis activity of trsa 
 is around twice the trans - sialylation rate catalyzed 
 by tcts .
our theoretical data also support 
 these conclusions since the energy barrier of the trsa hydrolysis 
 is 4 kcal / mol lower than tcts trans - sialylation and 
 the final products of the reaction have similar energies .
finally , the energy decomposition 
 method was employed to identify the influence of individual active 
 site residues on the energy barrier of the hydrolysis of the ciwat ( figure 7 ) .
relative stabilization 
 pattern of the most relevant active site 
 residues on the tswat considering the ciwat as 
 reference .
as expected , all of the residues 
 that showed a significant effect 
 on the energy barrier had an opposite behavior to the one observed 
 in our previous work during the formation of the cilac from 
 the mclac .
the residues that 
 stabilized the ts in one reaction are responsible for a destabilizing 
 effect on the other and vice versa .
this is due to the fact that the 
 nucleophile and the leaving group are inverted in both reactions . 
 in the former ,
the nucleophilic group was the tyr342 hydroxyl and 
 the leaving group was the lactose molecule . in the present study , 
 a water molecule represents the nucleophile while tyr342 is the leaving 
 group .
two out of the three arginines ( arg245 and arg314 ) of the arginine 
 triad together with glu362 considerably contribute to stabilize the 
 tswat whereas arg35 has a destabilizing effect .
two additional 
 arginines , arg53 and arg251 , also display an important effect , increasing 
 and lowering the energy barrier , respectively .
in addition , asp247 , 
 which interacts with arg245 , has a significant destabilizing effect . 

all of those arginines remain hydrogen - bonded to the carboxylate group 
 of the sialic acid except for arg53 , which is interacting with asp59 . 

the stabilizing or destabilizing behaviors brought about by those 
 residues are common to both trsa and tcts although the effects are 
 larger in trsa , which can be explained considering the following facts . 

configurations of the mcwat complex of both trsa and tcts 
 are very similar , but this is not the case for the tswat structures . as it was mentioned , d2 is 
 shorter for trsa whereas d3 is larger . 

those differences account for a higher ionic character of both substrates 
 in trsa ( i.e. , the water molecule resembles more a hydroxyl group 
 and the sialic acid adopts a higher oxicarbenium character ) . as a 
 consequence ,
the interaction between the active site residues and 
 the enzyme substrates is stronger in trsa than in tcts which , in turn , 
 is seen as a larger stabilizing / destabilizing effect of the common 
 active site residues .
important effects are observed in other 
 residues from the catalytic 
 cleft , though the contributions to the stability of the tswat are not as large as the ones presented by the former arginines . 

interestingly , ser119 contributes to lowering the energy barrier in 
 trsa ( e = 3.0 kcal / mol ) while 
 the tyrosine shows the opposite effect in tcts ( e = + 3.7 kcal / mol ) .
this finding , together with the fact 
 that ser119 favors the solvent exposure of the catalytic cleft and 
 the stabilization of the water molecule in the active site of trsa , 
 helps to rationalize why trsa strictly displays hydrolase activity , 
 contrary to tcts .
moreover , when ser119 was mutated to tyrosine in 
 trsa , the sialidase activity was reduced by 50% . on the contrary , in tcts , the aromatic side chain of tyr119 , together 
 with trp312 , forms a hydrophobic pocket that excludes water from the 
 active site , hindering the hydrolysis 
 reaction on one hand and favoring the transfer activity on the other 
 by establishing stacking interactions with the incoming lactose .
the ciwat hydrolysis reactions catalyzed 
 by tcts and 
 trsa were simulated using qm / mm calculations combined with umbrella 
 sampling simulations .
the fes obtained showed a clear mfep between 
 reactants ( ciwat ) and products ( mcwat ) . in both 
 enzymes
the 
 free energy calculations presented here together with our previous 
 studies of the ts reactions allow us to obtain the following conclusions : 
 ( i ) the estimated reaction barrier for the hydrolysis of the ciwat by a water molecule catalyzed by trsa is 16.4 kcal / mol , 
 i.e. , 10 kcal / mol lower than the same reaction catalyzed by tcts ( 26.8 
 kcal / mol ) , and explains why trsa is a more efficient hydrolase than 
 tcts .
( ii ) in tcts , the energy barrier to accomplish the transfer 
 reaction is 6 kcal / mol lower compared to the sialidase reaction .
tcts 
 would then preferentially behave as a trans - sialidase 
 rather than a sialidase .
moreover , the free energy difference also 
 accounts for the establishment of a long - lived ci favoring the tcts trans - sialidase activity .
( iii ) in trsa , the hydrolysis 
 reaction barrier is 10 kcal / mol lower than the transfer reaction , 
 indicating that trsa would have a more important role as a sialidase .
in addition , the energy decomposition allowed us to identify that , 
 due to differential conformations , some residues ( particularly arg245 
 and arg314 ) show a distinct stabilization pattern on tswat in trsa and tcts . besides , the presence of ser119 in trsa slightly 
 stabilizes the tswat while tyr119 has the opposite effect 
 in tcts . besides the energetic dissimilarities shown above for 
 the catalytic 
 mechanism of tcts and trsa , other characteristics such as differential 
 exclusion of water molecules from the active sites , stability of the 
 active site residue conformations at the ci stage , and others aspects 

should also be considered to fully understand the distinctive properties 
 of these highly similar enzymes .
results presented here provide 
 new insights into the catalytic 
 mechanisms of tcts and trsa that complement our previous observations . 
 taken together , they help us to rationalize from different perspectives 
 why trsa behaves as a strict hydrolase whereas tcts preferentially 
 acts a trans - sialidase .
we hope this work can contribute 
 to a deeper understanding of enzyme catalysis and to the development 
 of new drugs leading to the inhibition of tcts toward a possible treatment 
 of the expanding chagas disease .
 chagas disease , also known 
 as american trypanosomiasis , 
 is a lethal , chronic disease that currently affects more than 10 million 
 people in central and south america . 
 the trans - sialidase 
 from trypanosoma cruzi ( t. cruzi , tcts ) is a crucial enzyme 
 for the survival of this parasite : sialic acids from the host are 
 transferred to the cell surface glycoproteins of the trypanosome , 
 thereby evading the host s immune system . on the other hand , 
 
 the sialidase of t. rangeli ( trsa ) , which shares 
 70% sequence identity with tcts , is a strict hydrolase and shows no trans - sialidase activity . 
 therefore , tcts and trsa represent 
 an excellent framework to understand how different catalytic activities 
 can be achieved with extremely similar structures . by means of combined 
 quantum mechanics  
 molecular mechanics ( qm / mm , scc - dftb / amberff99sb ) 
 calculations and umbrella sampling simulations , we investigated the 
 hydrolysis mechanisms of tcts and trsa and computed the free energy 
 profiles of these reactions . 
 the results , together with our previous 
 computational investigations , are able to explain the catalytic mechanism 
 of sialidases and describe how subtle differences in the active site 
 make trsa a strict hydrolase and tcts a more efficient trans - sialidase . 
Introduction
Methods
Results and Discussion
Conclusion