Beaty N B, Lane M D
J Biol Chem. 1983 Nov 10;258(21):13043-50.
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.
比较了柠檬酸诱导的450,000道尔顿原聚体形式的乙酰辅酶A羧化酶激活和聚合（形成细丝）的动力学，以评估这两个过程的协同性。采用快速淬灭技术来测量柠檬酸对羧化酶催化反应的激活时间进程。当在快速淬灭装置中用乙酰辅酶A启动稳态周转反应之前，将酶与柠檬酸预孵育时，从混合时刻起观察到的乙酰辅酶A羧化速率明显呈线性。然而，当将酶与柠檬酸混合以启动反应时，在达到稳态羧化速率的过程中出现了一个滞后（t1/2 = 0.7秒）。这个滞后在230倍的酶浓度范围内与酶浓度无关，并且对柠檬酸浓度的依赖性很小。在相同的酶浓度范围内，通过直角光散射测定的羧化酶原聚体的聚合以单个原聚体激活步骤预测的方式依赖于酶浓度，随后是活性原聚体的限速二聚化和随后的聚合。基于这些结果，可以得出结论，催化激活和羧化酶原聚体的聚合不是协同的。此外，在所有条件下，导致活性原聚体形成的羧化激活都比聚合快，因此先于聚合。还表明，柠檬酸的激活常数（Kact）会因反应产物丙二酰辅酶A的积累而以可预测的方式改变，Kact随着丙二酰辅酶A浓度的增加而增加。提供了额外的证据表明，在这些条件下，Kact的这种变化不是由酶的自磷酸化引起的，并且磷酸化不影响上述激活机制。
