Electron Paramagnetic Resonance, 1H, and 13C Nuclear Magnetic Resonance Studies of the Interaction of Manganese and Bicarbonate with Ribulose 1,5-Diphosphate Carboxylase

摘要: Abstract The binding of manganese by ribulose 1,5-diphosphate carboxylase is dependent upon the level bicarbonate in solution. Saturating levels (50 mm) create one tight Mn2+-binding site (Kd = 10 µm) per 70,000 dalton subunit enzyme. dissociation constant enzyme-Mn2+ complex becomes 64-fold smaller when solution raised from l0.5 mm to 50 mm. Binding Mn2+ enzyme (in presence saturating bicarbonate) causes a 20-fold enhancement effect on longitudinal relaxation rate (1/T1) water. due enzyme-bound at low only 14-fold. Titration an with yields (7 which reasonable agreement reported Km values 25 mm). complexes (at varying can be completely abolished addition carboxyribitol diphosphate, analog presumed carboxylated intermediate reaction. A results there little access water protons enzymebound Mn2+. Observation time-dependent decrease 1/T1 diphosphate and nonclassical titration curves suggests conformation change 13C nuclear magnetic resonance shows that carbon enhanced more than 3-fold over nonenzyme control, suggesting very close bound bicarbonate. From measurements atom correlation time estimated proton rates same complex, Mn2+-carbon distance 5.4 calculated. This value too great for inner coordination sphere Mn2+, but consistent second complex. Carboxyribitol abolishes all effects 13C-relaxation rates, displacement HCO3- active NMR data are quaternary enzyme-manganese-d-ribulose 1,5-diphosphate-CO2 d-ribulose CO2 temperature dependence transverse exchange environment 3 x 104 s-1 energy activation, Ea 7.3 Cal mole.