Ribulose-1,5-biphosphate carboxylase. Evidence in support of the existence of distinct CO2 activator and CO2 substrate sites.

ABSTRACT

Ribulose-1,5-biphosphate carboxylase forms a complex with Mn2+ and CO2 that exhibits a considerably enhanced water proton relaxation rate. This effect is diminished upon interaction of the complex with the substrate, ribulose biphosphate, or with the competitive inhibitor, ribitol 1,5-biphosphate (Ki=0.55 mM). Included among the several mechanisms which can explain these observations is the possibility that a slow exchange of metal ligands occurs. Attempts at testing the feasibility of a slow exchange mechanism led to demonstration of a stable complex of enzyme with CO2 in the presence of metal and carboxyribitol biphosphate (CRBP), an analog of the hypothetical transition state intermediate. The complex formed upon mixing these components is stable to Sephadex G-75 chromatography and contains a nonexchangeable [14C]O2 bound stoichiometrically with respect to enzyme active sites. Mg2+ or Mn2+ can be used to form the E.M.[14C]O2.CRBP complex, which is stable with respect to CO2 exchange until the enzyme is denatured with sodium dodecyl sulfate. If the tight, functionally irreversible binding of the transition state analog is due to its occupancy of ribulose biphosphate and substrate CO2 sites, then simultaneous stoichiometric binding of [14C]O2 to enzyme indicates that 2 CO2 molecules participate in photosynthetic carbon fixation.