AVIDIN IS A SLOW-BINDING INHIBITOR OF PYRUVATE-CARBOXYLASE
AVIDIN IS A SLOW-BINDING INHIBITOR OF PYRUVATE-CARBOXYLASE
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DOI：
10.1021/bi00257a018
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发表时间：
1982-01-01
期刊：
影响因子：
2.9
通讯作者：
KEECH, DB
中科院分区：
文献类型：
--
作者：
DUGGLEBY, RG;ATTWOOD, PV;KEECH, DB
在抗生物素蛋白存在下，丙酮酸羧化酶反应的进展曲线显示在几分钟内反应速率显著降低。这些曲线定量地描述了一个方程，其中酶的催化速率经历了一个一级衰减，从一个高的初始速度的最终速度接近零。反应的初速度与亲和素浓度无关，而衰减常数随亲和素浓度的增加而线性增加。这些发现与游离酶和酶-亲和素复合物之间缓慢平衡的模型一致。涉及游离酶或酶-抗生物素蛋白复合物的缓慢异构化的替代模型与数据不一致。从衰减常数对抗生物素蛋白浓度曲线的斜率，计算出酶-抗生物素蛋白复合物形成的速率常数为（1.42 ± 1.42）。0.09）×105 M-1 s-1的酶分离自鸡肝。该图的截距等于酶-抗生物素蛋白复合物解离的速率常数，该截距未被数据很好地限定，但估计小于2 × 10 - 4。10-3 s-1条件下鉴于这种不确定性，使用了替代程序来获得更可靠的值。该方法包括在酶-抗生物素蛋白复合物与生物素温育后再生酶活性，并得到（1.45 ± 1.55）的值。0.13）×10-5 s-1为解离速率常数。酶-抗生物素蛋白复合物发生反应，该反应使酶的抑制不可逆，并且发生的速率比抗生物素蛋白从酶上解离的速率高5倍。这一观察结果解释了广泛持有的观点，即抗生物素蛋白抑制是不可逆的。
In the presence of avidin, progress curves for the pyruvate carboxylase reaction show a marked decrease in the rate of reaction over a period of several minutes. These curves are quantitatively described by an equation in which the rate of catalysis by the enzyme undergoes a first-order decay from a high initial velocity to a final velocity which is close to zero. The initial velocity of the reaction is independent of avidin concentration while the decay constant increases linearly with increasing concentrations of avidin. These findings are in agreement with a model in which there is slow equilibration between free enzyme and the enzyme-avidin complex. Alternative models involving slow isomerization of the free enzyme or of an enzyme-avidin complex are not consistent with the data. From the slope of the decay constant vs. avidin concentration plot, the rate constant for formation of the enzyme-avidin complex was calculated to be (1.42 .+-. 0.09) .times. 105 M-1 s-1 for the enzyme isolated from chicken liver. The intercept of this plot, which is equal to the rate constant for dissociation of the enzyme-avidin complex, was not well-defined by the data but was estimated to be less than 2 .times. 10-3 s-1. In view of this uncertainty, an alternative procedure was used to obtain a more reliable value. This procedure involved following the regeneration of enzymic activity upon incubation of the enzyme-avidin complex with biotin and yielded a value of (1.45 .+-. 0.13) .times. 10-5 s-1 for the dissociation rate constant. The enzyme-avidin complex undergoes a reaction which renders the inhibition of the enzyme irreversible and which occurs at a rate that is 5 times greater than the rate of dissociation of the avidin from the enzyme. This observation accounts for the widely held view that avidin inhibition is irreversible.