Abstract
Citrate, an allosteric activator of acetyl-CoA carboxylase, induces polymerization of an inactive protomeric form of the enzyme into an active filamentous form composed of 10-20 protomers. The light-scattering properties of the carboxylase were used to study the kinetics of its polymerization and depolymerization. From stopped flow kinetic studies, we have established that polymerization is a second order process, with a second order rate constant of 597,000 M-1 s-1. There appear to be two steps which limit polymerization of the inactive carboxylase protomer: 1) a rapid citrate-induced conformational change which is independent of enzyme concentration and leads to an active protomeric form of the enzyme (Beaty, N. B., and Lane, M. D. (1983) J. Biol. Chem. 258, 13043-13050, preceding paper) and 2) the dimerization of the active protomer, which constitutes the first step of polymerization and is enzyme concentration-dependent. Dimerization is the rate-limiting step of acetyl-CoA carboxylase polymerization. Depolymerization of fully polymerized acetyl-CoA carboxylase is caused by malonyl-CoA, ATP X Mg, and Mg2+. Both malonyl-CoA and ATP X Mg (and HCO-3) compete with citrate in the maintenance of a given state of the protomer-polymer equilibrium apparently by carboxylating the enzyme to form enzyme-biotin-CO-2 which destablizes the polymeric form. Free citrate is the species responsible for polymerizing the enzyme and Mg2+ causes depolymerization of the enzyme by lowering the concentration of free citrate.
Highlights
From stopped flow kinetic studiesw, e have estab- and in assay reaction mixture minus citrate), bothprocesses lished that polymerization is a second order process, followed first order kinetics and occurred at the same rate with a second order rate constant of 597,000 M-’ s-’. ( t I l 2= 9 min)
In the present instate of the protomer-polymer equilibrium apparently vestigation, we have followed the kinetics of polymerization by carboxylating the enzyme to form enzyme-biotin- of acetyl-coA carboxylase to its filamentousform by stopped
Our results indicate that is the speciesresponsible for polymerizing the enzyme two stepslimit polymerization of the inactivecarboxylase and Mg2+causes depolymerization of the enzyme by lowering theconcentration of free citrate
Summary
Viscosity (Moss and Lane, 1972a) and the polymerization of Light-scattering and Stopped Flow Measurements-Kinetic data the enzyme into filamentsof up to 0.5 pm in length (Lane et were collected on a Durrum stopped flow apparatus connected to a al., 1974; Kleinschmidt et al, 1969; Lane et al, 1975). 1)as itdid with enzyme concentrations below 0.4 mg/ml These results and those presented in the preceding paper on the citrate-induced activation of the carboxylase (Beaty and Lane,1983) are most consistent with tfhoellowing kinetic pancy is the per cent of total change based on anend point determined model. The preparation was cleared of any debris by centrifugation at 30,000X g for 15 min and the actual enzyme concentration was determined from the 280 nm absorbance be 0.78 s-' (Beaty and Lane, 1983). Since dimerization is dependent on the concentration of activated protomer (E*-Cit), which is produced from the initial enzyme concentration by way of the citrate-induced conformational change, raotfepolymerization using the relationship Aeao,", X 0.86 = 1 mg/ml (Moss and Lane, 1972a). Theinitialevents of polymerization areratelimited by citrateactivation,thelinear second orderplots
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