Beaty N B, Lane M D
Ann N Y Acad Sci. 1985;447:23-37. doi: 10.1111/j.1749-6632.1985.tb18423.x.
The kinetic time course of citrate-induced activation and polymerization (into filaments) of the protomeric form of acetyl-CoA carboxylase were compared to assess the concertedness of the two processes. Rapid-quench techniques were employed to measure the kinetics of activation of the carboxylase-catalyzed reaction by citrate. 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. 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 that limit polymerization of the inactive carboxylase protomer: a rapid citrate-induced conformational change, which is independent of enzyme concentration and leads to an active protomeric form of the enzyme and 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. On the basis 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 malonyl-CoA concentration. Depolymerization of fully polymerized acetyl-CoA carboxylase is caused by malonyl-CoA or ATP.Mg (and HCO3-). Both malonyl-CoA and ATP.Mg (and HCO3-) compete with citrate in the maintenance of a given state of the protomer-polymer equilibrium apparently by carboxylating the enzyme to form enzyme-biotin CO2- which destabilizes the polymeric form.
比较了柠檬酸诱导的乙酰辅酶A羧化酶原聚体形式的激活和聚合（形成细丝）的动力学时间进程，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动力学。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延迟（t1/2 = 0.7秒）。这个延迟在230倍的酶浓度范围内与酶浓度无关，并且仅略微依赖于柠檬酸浓度。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是酶浓度依赖性的，其方式由单个原聚体激活步骤预测，随后是活性原聚体的限速二聚化以及随后的聚合。聚合是一个二级过程，二级速率常数为597,000 M-1 s-1。似乎有两个步骤限制了无活性羧化酶原聚体的聚合：一个是柠檬酸诱导的快速构象变化，它与酶浓度无关，导致酶的活性原聚体形式，以及活性原聚体的二聚化，这是聚合的第一步，并且是酶浓度依赖性的。二聚化是乙酰辅酶A羧化酶聚合的限速步骤。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随丙二酰辅酶A浓度增加。完全聚合的乙酰辅酶A羧化酶的解聚是由丙二酰辅酶A或ATP·Mg（以及HCO3-）引起的。丙二酰辅酶A和ATP·Mg（以及HCO3-）显然通过使酶羧化形成酶 - 生物素CO2-来破坏聚合物形式，从而在维持原聚体 - 聚合物平衡的给定状态方面与柠檬酸竞争。
