果蝇胚胎中3':5'-环磷酸腺苷结合及蛋白激酶激活模式的动力学研究。

A kinetic study of cyclic adenosine 3':5'-monophosphate binding and mode of activation of protein kinase from Drosophila melanogaster embryos.

作者信息

Tsuzuki J, Kiger J A

出版信息

Biochemistry. 1978 Jul 25;17(15):2961-70. doi: 10.1021/bi00608a004.

PMID:212097

Abstract

Cyclic AMP-dependent protein kinase and its regulatory subunit were isolated from Drosophila melanogaster embryos. The profiles of cyclic AMP binding by these proteins were significantly different. In order to explain such a difference and to find the mode of enzyme activation by cyclic AMP, a kinetic study of cyclic AMP binding was carried out. First, the association rate constant k1 and dissociation rate constant k-1 in the cyclic AMP-regulatory subunit interaction at 0 degrees C were estimated to be 2.3 X 10(6)M-1s-1 and 1.1 X 10(-3)s-1, respectively. Secondly, the three possible modes of enzyme activation by cyclic AMP were mathematically considered and could be described by a unique formula: r=APt + BQt (A + B=1) in which the parameters A, B, P, and Q are equivalent to rate constants in the sense that the rate constants are simply expressed by these parameters. Thirdly, the values of the parameters and subsequently the values of rate constants involved in the possible mechanisms were evaluated using a curve-fitting technique and compared with experimental observation. It was then found that the following mechanism was the only one which fitted the experimental observations. Namely, RC + L k3 equilibrium k-3 LRC k4 equilibrium k-4 RL + C where R, C, and L represent the regulatory and catalytic subunits and cyclic AMP as a ligand. Thus, our results indicate that in the presence of cyclic AMP the active enzyme (C) is released from a ternary intermediate which is the primary product of the cyclic AMP-holoenzyme interaction. The estimated values of the rate constants are: k3=3.5 X 10(6)M-1s-1;k-3=7.3 X 10(-1)s-1;and k4=3.8 X 10(-2)s. These estimates indicate that the reaction LRC leads to RL + C is relatively slow and limits the rate of the overall reaction. By comparing k-3 and k4, it is apparent that a large part of newly formed ternary intermediate reverts to the holoenzyme.

摘要

环磷酸腺苷（cAMP）依赖性蛋白激酶及其调节亚基是从黑腹果蝇胚胎中分离出来的。这些蛋白质与环磷酸腺苷的结合情况明显不同。为了解释这种差异并找到环磷酸腺苷激活该酶的模式，我们对环磷酸腺苷的结合进行了动力学研究。首先，在0℃下，环磷酸腺苷与调节亚基相互作用的缔合速率常数k1和解离速率常数k-1分别估计为2.3×10⁶M⁻¹s⁻¹和1.1×10⁻³s⁻¹。其次，从数学上考虑了环磷酸腺苷激活酶的三种可能模式，并且可以用一个独特的公式来描述：r = APt + BQt（A + B = 1），其中参数A、B、P和Q在某种意义上等同于速率常数，即速率常数可以简单地用这些参数表示。第三，使用曲线拟合技术评估了可能机制中参数的值以及随后涉及的速率常数的值，并与实验观察结果进行了比较。结果发现，以下机制是唯一符合实验观察结果的机制。即，RC + L⇌k₃k⁻₃LRC⇌k₄k⁻₄RL + C，其中R、C和L分别代表调节亚基、催化亚基和作为配体的环磷酸腺苷。因此，我们的结果表明，在存在环磷酸腺苷的情况下，活性酶（C）从三元中间体中释放出来，该三元中间体是环磷酸腺苷 - 全酶相互作用的主要产物。速率常数的估计值为：k₃ = 3.5×10⁶M⁻¹s⁻¹；k⁻₃ = 7.3×10⁻¹s⁻¹；k₄ = 3.8×10⁻²s。这些估计表明反应LRC→RL + C相对较慢，限制了整个反应的速率。通过比较k⁻₃和k₄，很明显，大部分新形成的三元中间体又变回了全酶。