The determination of enzyme dissociation constants
The determination of enzyme dissociation constants
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DOI：
10.1021/ja01318a036
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发表时间：
1934-01-01
影响因子：
15
通讯作者：
Burk, D
中科院分区：
文献类型：
--
作者：
Lineweaver, H;Burk, D
开发了包括恒斜率和直线外推的图解方法，用于测试和解释动力学数据，并在数据与指定的机理一致时确定酶-底物和酶-抑制剂化合物的离解常数和其他相关常数。对蔗糖酶、棉籽酶、淀粉酶、柠檬酸脱氢酶、过氧化氢酶、加氧酶、酯酶和脂肪酶进行了代表性的分析，包括底物激活、底物抑制、一般竞争和非竞争抑制、稳态和各种级数的反应。观察到的速度(V)与底物浓度(S)的倒数的曲线图在最简单的情况下(例如转化酶、淀粉酶)产生了一条直线，其斜率和二次截距得到Ks(米氏解离常数)和Vmax(理论最大速度)。在竞争性抑制剂的存在下，斜率增加，但截距不变。对于非竞争性抑制剂，截距也被提高了。所描述的各种方法适用于一般的化学催化，无论是均相还是非均相，并且与迄今采用的开发较少的方法相比，具有许多有用和方便的优点。
Graphical methods involving constant slopes and straight line extrapolations were developed for testing and interpreting kinetic data, and for determining dissociation constants of enzyme-substrate and enzyme-inhibitor compounds and other related constants when the data are consistent with an assigned mechanism. Representative analyses are given for invertase, raffinase, amylase, citric dehy-drogenase, catalase, oxygenase, esterase and lipase, involving substrate activation, substrate inhibition, general competitive and non-competitive inhibition, steady states and reactions of various orders. A plot of the reciprocal of the observed velocity (V) against the reciprocal of the substrate concentration (S) yielded in the simplest case (e.g., invertase, amylase) a straight line whose slope and ondinate intercept yielded Ks (Michaelis dissociation constant) and Vmax (theoretical maximum velocity). In the presence of competitive inhibitors the slope was increased but the intercept was unchanged. With non-competitive inhibitors the intercept also was raised. The various methods described are applicable to general chemical catalysis, homogeneous or heterogeneous, and possess many advantages of usefulness and convenience over less extensively developed methods employed heretofore.