Picomolar Inhibitors as Transition-State Probes of 5′-Methylthioadenosine Nucleosidases

Abstract

Transition states can be predicted from an enzyme's affinity to related transition-state analogues. 5'-Methylthioadenosine nucleosidases (MTANs) are involved in bacterial quorum sensing pathways and thus are targets for antibacterial drug design. The transition-state characteristics of six MTANs are compared by analyzing dissociation constants (K(d)) with a small array of representative transition-state analogues. These inhibitors mimic early or late dissociative transition states with K(d) values in the picomolar range. Our results indicate that the K(d) ratio for mimics of early and late transition states are useful in distinguishing between these states. By this criterion, the transition states of Neisseria meningitides and Helicobacter pylori MTANs are early dissociative, whereas Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, and Klebsiella pneumoniae MTANs have late dissociative characters. This conclusion is confirmed independently by the characteristic [1'- (3)H] and [1'- (14)C] kinetic isotope effects (KIEs) of these enzymes. Large [1'- (3)H] and unity [1'- (14)C] KIEs are observed for late dissociative transition states, whereas early dissociative states showed close-to-unity [1'- (3)H] and significant [1'- (14)C] KIEs. K d values of various MTANs for individual transition-state analogues provide tentative information about transition-state structures due to varying catalytic efficiencies of enzymes. Comparing K d ratios for mimics of early and late transition states removes limitations inherent to the enzyme and provides a better predictive tool in discriminating between possible transition-state structures.

Neutron structures of the Helicobacter pylori 5′-methylthioadenosine nucleosidase highlight proton sharing and protonation states

“…It was shown that the K d for p-ClPh-Thio-DADMe-ImmA was 570 pM for HpMTAN, an affinity roughly 100-fold higher than that of the ImmA analog that was demonstrated to have a K d of 40 nM (16). The differences in the affinities of the transition state analogs to structurally similar homologs of MTAN have been studied previously using computational techniques (38,39).…”

“…The DADMe-ImmA analogs represent a late dissociative transition state by containing a cationic nitrogen atom in the pyrrolidine moiety that represents a fully formed carbocation as well as having an elongated distance of 2.5 Å between the nonhydrolyzable adenine mimic and pyrrolidine moiety. Although previously published kinetic data identify HpMTAN as forming an early dissociative transition state during catalysis, the most potent transition-state analogs are DADMeImmA derivatives, which exhibit K d values in the picomolar range (16,17).…”

“…Data deposition: Crystallography, atomic coordinates, and structure factors reported in this paper have been deposited in the Protein Data Bank (PDB ID codes 5CCD, 5K1Z, 5JPC, 5CCE, and 5KB3). (DADMe-ImmA) derivatives (16). These compounds are protonated at the N7 position of the adenine moiety but differ significantly in the ribose mimic and vary in the distance between the ribose-and adenine-mimicking moieties.…”

Inhibition and Structure of Toxoplasma gondii Purine Nucleoside Phosphorylase

“…Transition-state analogues that mimic the specific geometry and electrostatic features of a molecule can be used to predict the position of the transition state in the reaction coordinates (29). The dissociation constants of an array of transition-state analogue inhibitors were measured to explore the specificity of TgPNP.…”

“…Transition-state structures can be predicted based upon affinity to related transitionstate analogues. The transition-state characteristics are compared by analyzing dissociation constants (K i ) of an array of representative transition-state analogues (29). These inhibitors mimic known early or late dissociative transition states.…”