In the previous section, we have seen that H138 is crucial as a base for the 'kinase activity'. A study using site-directed mutagenesis indicates that a mutation of H138L reduces the native 'kinase activity' while at the same time, it causes the emergence of a secondary function as an ATPase activity. To study the mechanism of the function of the promiscuity due to a single site mutation, we performed an MD simulation of the H138L mutant of homoserine kinase complexed with its native substrate and compared it with the WT simulation. In the MD simulations of the WT complex, we see that δ-H of H138 forms a strong hydrogen bond with O1 of carboxylate of D23. At the This method is statistically preferable to a stochastically chosen snapshot. same time, the second oxygen O2 of carboxylate of D23 forms another hydrogen bond with the hydrogen of the guanidium group of R187. In this way, the D23, H138, and R187 triad and the substrate L-homoserine form a closed door that blocks the access of the water channel to the ATP (see Figure ). Since the ATPase activity is a water-mediated reaction where ATP gets converted to ADP and inorganic phosphate, such blockage of the water access inhibits the ATPase activity, and in turn, we see no ATPase activity in the WT HSK. Interestingly, in the H138L mutant, the bridging due to H138-D23 interaction is lost, and it opens the water doorway connecting through the ATP. Additionally, Leucine being hydrophobic moves away from the channel and creates extra room for the easy passage of the water molecule (see Figure ). This is well supported by distance plots of H138/L138 with D23 and D23 and R187 in WT/mutant simulations (see Figure ) and increased water population within 3Å of the active site as shown in Figure .