Characterization of Catalysis and Binding in MTANs

Abstract

5'-Methylthioadenosine/S-adenosyl homocysteine nucleosidase (MTAN) is a bacterial enzyme involved in quorum sensing in Gram negative bacteria and in menaquinone synthesis in several gastrointestinal pathogens. MTAN is not found in humans and is a candidate for antibacterial drug design efforts. MTAN inhibition prevents bacterial quorum sensing resulting in the uncoordinated expression of virulence genes. Drug design efforts with MTAN as the target have utilized transition state analysis and have yielded transition state analogues with femtomolar dissociation constants. E. coli (Ec), S. enterica (Se) and V. cholerae (Vc) MTANs have late dissociative transition states, showing little participation from either the attacking nucleophile or the purine leaving group. The active sites of Ec, Se- and Vc- MTANs have near-identical protein structural folds and similar active site arrangements, yet the affinities for transition state analogues vary between enzymes.;We examined whether differences in the active site or protein dynamic architecture are responsible for these different catalytic properties. The thermodynamic signature for the binding of transition state analogues to Ec- and Se- MTANs revealed large favorable enthalpies and smaller, mostly favorable entropic factors. VcMTAN shows smaller enthalpy and larger favorable entropy factors than Ec- and Se- MTANs. Variations in transition state analogue affinities among the MTANs are due to overall protein architecture rather than the first sphere catalytic residues. Catalytic non-equivalence and one-site inhibition were established between monomers of some homodimers.;The thermodynamic signatures for transition state analogue binding to VcMTAN and EcMTAN are markedly different, despite small variations in the active site structure and hydrogen bond network between enzyme and inhibitor. The two amino acids distinguishing the active sites of VcMTAN from EcMTAN were altered to determine if they are responsible for the different MTAN thermodynamic properties. Mutagenized VcMTAN, with an identical active site to EcMTAN, exhibited kinetic and thermodynamic properties more unlike EcMTAN, compared with the native VcMTAN. The presence of a single conservative mutation [valine-153-isoleucine] resulted in full catalytic inhibition; only one catalytic site binds inhibitor. Kinetic and thermodynamic analyses of native and mutant MTANs establish that the protein architecture outside the catalytic site determines catalytic site function.;A `heavy' E. coli MTAN was expressed in which the non-exchangeable hydrogens, carbons and nitrogens were substituted with the heavy isotopes. We probed the involvement of vibrational dynamics in formation of the transition state by comparing the kinetic constants generated by the heavy and light (atoms with natural isotopic abundance) EcMTANs. The bond vibrational frequencies in the heavy EcMTAN are decreased compared to the light ECMTAN. Preliminary data suggest that the altered bond vibrational frequency in the heavy EcMTAN affects barrier crossing, albeit differently from other heavy enzymes, suggesting faster on-enzyme chemistry in heavy EcMTAN.;The tight binding of transition state analogues in Ec-, Vc, and Se- MTANs is due to both favorable enthalpic and entropic binding components. The whole protein architecture in addition to the immediate catalytic residues is involved in binding transition state analogues in VcMTAN. A single residue -- Val153, disrupts communication between monomers of the dimeric VcMTAN. This work advances the understanding of interactions between MTANs and its transition state analogues.