Kinetics of activation of acetyl-CoA carboxylase by citrate. Relationship to the rate of polymerization of the enzyme.

Abstract

The kinetics of citrate-induced activation and polymerization (into filaments) of the 450,000-dalton protomeric form of acetyl-CoA carboxylase were compared to assess the concertedness of the two processes. Rapid-quench techniques were employed to measure the time course of activation by citrate of the carboxylase-catalyzed reaction. When enzyme was preincubated with citrate prior to initiating the steady state turnover reaction with acetyl-CoA in the rapid-quench device, the observed rate of carboxylation of acetyl-CoA was apparently linear from the moment of mixing. However, when enzyme was mixed with citrate to initiate the reaction, a lag (t1/2 = 0.7 s) occurred in the approach to steady state carboxylation rate. This lag was independent of enzyme concentration over a 230-fold range and was marginally dependent upon citrate concentration. Over the same range of enzyme concentration, polymerization of carboxylase protomers, as determined by right angle light scattering, was enzyme concentration-dependent in a manner predicted by a single protomer activation step, followed by a rate-limiting dimerization of active protomer and subsequent polymerization. Based on these results, it is concluded that activation of catalysis and the polymerization of carboxylase protomers are not concerted. Furthermore, activation of carboxylation leading to the formation of an active protomer was faster than polymerization under all conditions, and therefore precedes polymerization. It was also shown that the activation constant (Kact) for citrate is altered in a predictable manner by the accumulation of the reaction product, malonyl-CoA, the Kact increasing with increasing malonyl-CoA concentration. Additional evidence is presented indicating that this change in Kact was not caused by autophosphorylation of the enzyme under these conditions and that phosphorylation does not affect the mechanism of activation elucidated above.