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