Ion coordinate defined as z 3 
d5 
zd6 {d7, where d5 is the distance between O4FADH and the proton being transferred, d6 is the distance between C1XGAL and O5XGAL and d7 is the distance between the proton and O5XGAL. Coordinate z 3 was sampled from 0.82 to 4.18 A. We also analysed the possibility of a direct proton transfer from N5FADH to O5XGAL. In order to do that we defined a reaction coordinate z’3 d’5 zd’6 {d’7, where d’5 is the N5FADH-H distance, d’6 the one between O5XGAL and C1XGAL and d’7 the O5XGAL-H distance. This coordinate was sampled from 21.85 to 0.71 A. In agreement with previous results of Huang et. al. we found that this direct proton transfer is very unlikely since it has a barrier significantly higher than the alternative path. Stage 3: Formation of the flavin-Galf adduct. This stage also occurs in two steps. First, the hydrogen attached to O4XGAL is transferred to O4FADH while a bond between O4XGAL and C1XGAL is formed. The reaction coordinate for this step was defined as z 4 d8 {d9 {d10, where d8 is the distance between the H atom being transferred and O4XGAL, d9 the distance between the H atom and O4FADH and d10 is the distance between O4XGAL and the C1XGAL. Coordinate z 4 was sampled from 24.85 to 21.01 A. The following step consists of a proton transfer from O4FADH to N5FADH. This can be seen as the reverse of step 2, except for the fact that galactose is now in the furanose form. Therefore, the reaction coordinate was defined as the reverse of step 2 and it was scanned from 21.63 to 1.65 A. Stage 4: Formation of UDP-Galf. This last step corresponds to the breakage of the bond between FADH2 and Galf along with the formation of a bond between Galf and UDP. Cambinol cost Galactopyranose/Galactofuranose Tautomerization in Trypanosoma cruzi Since this process is analogous to step 1 but occurs in reverse sense we defined z 6 d1 {d2 {z 1 and scanned it from 21.98 to 1.38 A. Energy decomposition An energy decomposition analysis was performed to evaluate how the active site residues stabilize or destabilize the transition states of the successive steps with respect to their correspondent reactants. Different variations of this idea have been implemented to study enzymatic reactions. In this case we followed the approach recently employed to compare the catalytic mechanisms of T. cruzi transialidase and T. rangeli sialidase. Since the approach has been discussed in detail elsewhere we only present here the most relevant equations. PubMed ID:http://jpet.aspetjournals.org/content/124/1/16 In the QM/MM study of an enzymatic reaction the influence of the classical environment on the activation energy of a given step, env DDERTS, can be evaluated as, QM env DDERTS DERTS {DERTS, QM=MM under analysis were His62, Val63, Arg176, Asn201, Tyr317, Z-IETD-FMK manufacturer Arg327, Tyr395, Arg423, Tyr429 and Asn433. i The DERTS computed in this way measures the difference between the actual barrier to reaction and the barrier that would be observed if the interaction between the side chain of residue i and the QM subsystem were turned off. Because of this, neither can it provide information about the effect of the backbone atoms or the effect of Gly residues. Moreover, since no dynamics is run i when the i-th residue is replaced by Gly, DERTS does not take into account dynamic effects arising from changes in the conformational freedom of the enzyme upon replacement. Finally i we note that positive/negative values of DERTS provide a strong indication about a deleterious/beneficial effect of residue i for the reaction step und.Ion coordinate defined as z 3 d5 zd6 {d7, where d5 is the distance between O4FADH and the proton being transferred, d6 is the distance between C1XGAL and O5XGAL and d7 is the distance between the proton and O5XGAL. Coordinate z 3 was sampled from 0.82 to 4.18 A. We also analysed the possibility of a direct proton transfer from N5FADH to O5XGAL. In order to do that we defined a reaction coordinate z’3 d’5 zd’6 {d’7, where d’5 is the N5FADH-H distance, d’6 the one between O5XGAL and C1XGAL and d’7 the O5XGAL-H distance. This coordinate was sampled from 21.85 to 0.71 A. In agreement with previous results of Huang et. al. we found that this direct proton transfer is very unlikely since it has a barrier significantly higher than the alternative path. Stage 3: Formation of the flavin-Galf adduct. This stage also occurs in two steps. First, the hydrogen attached to O4XGAL is transferred to O4FADH while a bond between O4XGAL and C1XGAL is formed. The reaction coordinate for this step was defined as z 4 d8 {d9 {d10, where d8 is the distance between the H atom being transferred and O4XGAL, d9 the distance between the H atom and O4FADH and d10 is the distance between O4XGAL and the C1XGAL. Coordinate z 4 was sampled from 24.85 to 21.01 A. The following step consists of a proton transfer from O4FADH to N5FADH. This can be seen as the reverse of step 2, except for the fact that galactose is now in the furanose form. Therefore, the reaction coordinate was defined as the reverse of step 2 and it was scanned from 21.63 to 1.65 A. Stage 4: Formation of UDP-Galf. This last step corresponds to the breakage of the bond between FADH2 and Galf along with the formation of a bond between Galf and UDP. Galactopyranose/Galactofuranose Tautomerization in Trypanosoma cruzi Since this process is analogous to step 1 but occurs in reverse sense we defined z 6 d1 {d2 {z 1 and scanned it from 21.98 to 1.38 A. Energy decomposition An energy decomposition analysis was performed to evaluate how the active site residues stabilize or destabilize the transition states of the successive steps with respect to their correspondent reactants. Different variations of this idea have been implemented to study enzymatic reactions. In this case we followed the approach recently employed to compare the catalytic mechanisms of T. cruzi transialidase and T. rangeli sialidase. Since the approach has been discussed in detail elsewhere we only present here the most relevant equations. PubMed ID:http://jpet.aspetjournals.org/content/124/1/16 In the QM/MM study of an enzymatic reaction the influence of the classical environment on the activation energy of a given step, env DDERTS, can be evaluated as, QM env DDERTS DERTS {DERTS, QM=MM under analysis were His62, Val63, Arg176, Asn201, Tyr317, Arg327, Tyr395, Arg423, Tyr429 and Asn433. i The DERTS computed in this way measures the difference between the actual barrier to reaction and the barrier that would be observed if the interaction between the side chain of residue i and the QM subsystem were turned off. Because of this, neither can it provide information about the effect of the backbone atoms or the effect of Gly residues. Moreover, since no dynamics is run i when the i-th residue is replaced by Gly, DERTS does not take into account dynamic effects arising from changes in the conformational freedom of the enzyme upon replacement. Finally i we note that positive/negative values of DERTS provide a strong indication about a deleterious/beneficial effect of residue i for the reaction step und.
