title: 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 ΔCpof 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 ΔCpfor binding. In addition, ΔCpvalues are not correlated with Kdvalues. 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 ΔCpfor 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.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Ross S
authors.initials: RS
authors.lastname: Firestone
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Cameron
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Jerome M
authors.initials: JM
authors.lastname: Karp
authors.affiliation: Faculty of Science and Engineering, Department of Biological Science, University of Waikato , Private Bag 3105, Hamilton, New Zealand.
authors.forename: Vickery L
authors.initials: VL
authors.lastname: Arcus
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Vern L
authors.identifier: 0000-0002-8056-1929
authors.initials: VL
authors.lastname: Schramm
mesh_terms: D002151:Calorimetry; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D011683:Purine-Nucleoside Phosphorylase / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D004791:Enzyme Inhibitors; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase