Transition states and transition state analogues for MTAP and MTANs

Abstract

The goal of the dissertation was to study the transition states (TS) of enzymes involved in the metabolism of 5'-methylthioadenosine using kinetic isotope effects (KIEs). Knowledge from the transition states is used to develop stable transition state analogue inhibitors. Kinetic and structural analyses with these inhibitors provide insight into catalysis. The transition states of three enzymes; human 5'-methylthioadenosine phosphorylase (human MTAP), and 5'-methylthioadenosine nucleosidase (MTAN) from E. coli and S. pneumoniae were explored. Human MTAP functions in polyamine and purine salvage pathways and is an anti-cancer target, whereas the bacterial enzymes function in quorum sensing pathways and are good antibacterial drug targets.;All three enzymes were found to have a dissociative SN1 TS. Instead of having an oxacarbenium ion TS, characteristic of dissociative S N1 transition states, computational matching of KIEs suggest one or more of the enzymes may have zwitterionic transition states, cationic at the anomeric carbon and anionic at the oxygen of 3'-OH. Further, the TS of S. pneumoniae MTAN is fully dissociative with no bond order to the anionic adenine leaving group or to the water nucleophile at the point of TS formation. The TS of human MTAP also has no bond order to the anionic leaving group, however, it has a significant participation by the phosphate nucleophile. The E. coli MTAN's TS has a neutral leaving group with minimal bond order to leaving group or water nucleophile. The conclusions of TS analyses were consistent with the kinetic studies using substrate analogues, mutational studies, and crystallographic studies of these enzymes with TS analogues. The KIEs at various positions on the substrate were investigated using advanced quantum chemistry methods.;A library of approximately fifty TS analogues was developed by collaborators and tested for their inhibitory potential. Immucillins were developed as analogues for associative SN1 TS whereas DADMe-Immucillins are mimics of more dissociative SN1 transition states. Inhibitors of the DADMe-Immucillin class were more powerful than Immucillins, consistent with TS analyses. At least ten inhibitors had femtomolar dissociation constants for E. coli MTAN. One inhibitor (p-chloro-phenylthio-DADMe-ImmA) binds with a Ki* of 47 femtomolar, 91 million times tighter than its substrate, S-adenosylhomocysteine. Crystallography of TS analogues with these enzymes provided further insight into the interactions responsible for their tight binding.