Heat capacity changes for transition-state analogue binding and catalysis with human 5 '-methylthioadenosine phosphorylase

Abstract

Human 5′-methylthioadenosine phosphorylase (MTAP) catalyzes the phosphorolysis of 5′-methylthioadenosine (MTA). Its action regulates cellular MTA and links polyamine synthesis to S-adenosylmethionine (AdoMet) salvage. Transition state analogues with picomolar dissociation constants bind to MTAP in an entropically driven process at physiological temperatures, suggesting increased hydrophobic character or dynamic structure for the complexes. Inhibitor binding exhibits a negative heat capacity change (−ΔCₚ), and thus the changes in enthalpy and entropy upon binding are strongly temperature-dependent. The ΔCₚ of inhibitor binding by isothermal titration calorimetry does not follow conventional trends and is contrary to that expected from the hydrophobic effect. Thus, ligands of increasing hydrophobicity bind with increasing values of ΔCₚ. Crystal structures of MTAP complexed to transition-state analogues MT-DADMe-ImmA, BT-DADMe-ImmA, PrT-ImmA, and a substrate analogue, MT-tubercidin, reveal similar active site contacts and overall protein structural parameters, despite large differences in ΔCₚ for binding. In addition, ΔCₚ values are not correlated with Kd values. Temperature dependence of presteady state kinetics revealed the chemical step for the MTAP reaction to have a negative heat capacity for transition state formation (−ΔCₚ‡). A comparison of the ΔCₚ‡ for MTAP presteady state chemistry and ΔCₚ for inhibitor binding revealed those transition-state analogues most structurally and thermodynamically similar to the transition state. Molecular dynamics simulations of MTAP apoenzyme and complexes with MT-DADMe-ImmA and MT-tubercidin show small, but increased dynamic motion in the inhibited complexes. Variable temperature CD spectroscopy studies for MTAP–inhibitor complexes indicate remarkable protein thermal stability (to Tₘ = 99 °C) in complexes with transition-state analogues.