Residence Times for Femtomolar and Picomolar Inhibitors of MTANs.

5'-Methylthioadenosine nucleosidases (MTANs) catalyze the hydrolysis of 5'-substituted adenosines to form adenine and 5-substituted ribose. Escherichia coli MTAN ( Ec MTAN) and Helicobacter pylori MTAN ( Hp MTAN) form late and early transition states, respectively. Transition state analogues designed for the late transition state bind with fM to pM affinity to both classes of MTANs. Here, we compare the residence times (off-rates) with the equilibrium dissociation constants for Hp MTAN and Ec MTAN, using five 5'-substituted DADMe-ImmA transition state analogues. The inhibitors dissociate orders of magnitude slower from Ec MTAN than from Hp MTAN. For example, the slowest release rate was observed for the Ec MTAN-HTDIA complex ( t 1/2 = 56 h), compared to a release rate of t 1/2 = 0.3 h for the same complex with Hp MTAN, despite similar structures and catalytic sites for these enzymes. Other inhibitors also reveal disconnects between residence times and equilibrium dissociation constants. Residence time is correlated with pharmacological efficacy; thus, experimental analyses of dissociation rates are useful to guide physiological function of tight-binding inhibitors. Steered molecular dynamics simulations for the dissociation of an inhibitor from both Ec MTAN and Hp MTAN provide atomic level mechanistic insight for the differences in dissociation kinetics and inhibitor residence times for these enzymes.