Heat Capacity Changes for Transition-State Analogue Binding and Catalysis with Human 5'-Methylthioadenosine Phosphorylase.
ACS Chem Biol
; 12(2): 464-473, 2017 02 17.
Article
en En
| MEDLINE
| ID: mdl-28026167
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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Purina-Nucleósido Fosforilasa
/
Calor
Límite:
Humans
Idioma:
En
Revista:
ACS Chem Biol
Año:
2017
Tipo del documento:
Article
País de afiliación:
Estados Unidos