Pre-steady-state and steady-state kinetic studies on transcription initiation catalyzed by T7 RNA polymerase and its active-site mutants K631R and Y639F.
Pre-steady-state and steady-state kinetic studies on transcription initiation catalyzed by T7 RNA polymerase and its active-site mutants K631R and Y639F.
复制标题
T7 RNA聚合酶及其活性位点突变体K631R和Y639F催化转录起始的前稳态和稳态动力学研究。
DOI：
10.1021/bi9805801
复制
发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Woody,RW
中科院分区：
文献类型：
--
作者：
Woody,AY;Osumi-Davis,PA;Hiremath,MM;Woody,RW
在不干扰酶- DNA解离或蛋白质再循环的条件下，研究了转录起始的动力学机制。稳态前动力学研究表明，野生型及其活性位点突变体K631R和Y639F的聚合速率常数分别为3.9、5.9和3.9 s-1，反聚合速率常数分别为3.2、2.1和2.8 s-1，进入的核苷酸在21°C下的解离常数分别为26、49和24 μM。结果表明，在该模型中，K631与传入底物的磷酸基相互作用。结合种的内部平衡常数接近统一，与其他磷酰转移酶的值一致。化学键形成的速率常数至少比产物解离的速率常数高50倍。产物释放速率常数k3与稳态速率相当，表明这三种酶的速率决定步骤可能是产物解离步骤。假设存在两种可能的构象E和E '处于快速平衡状态，以协调突变酶的充分活性与减少的爆发大小。两种形式都能形成四元配合物，但只有E型能催化磷酸二酯键的形成。催化活性E型的比例从野生型的100%到突变型K631R和Y639F的38%和32%不等。在进入延伸期后，E型成为所有三种酶的主导形式，导致野生型和Y639F突变体的延伸率相当。由于加工能力下降，K631R突变体的长转录本合成速率明显降低。
The kinetic mechanism of transcription initiation was studied under conditions that allow a single nucleotide addition to an initiating dinucleotide without interference of enzyme−DNA dissociation or protein recycling. Pre-steady-state kinetic studies have provided polymerization rate constants of 3.9, 5.9, and 3.9 s-1, reverse polymerization rate constants of 3.2, 2.1, and 2.8 s-1, and dissociation constants for the incoming nucleotide of 26, 49, and 24 μM at 21 °C, respectively, for the wild type and its active-site mutants K631R and Y639F. The results suggest a model in which K631 interacts with the phosphate group(s) of the incoming substrate. The internal equilibrium constants for the bound species are close to unity, consistent with the values for other phosphoryl transfer enzymes. The rate constants for chemical bond formation are at least 50 times higher than the rate constants for product dissociation. The product release rate constants,k3, are comparable to the steady-state rates, suggesting that the rate-determining step for all three enzymes may be a product dissociation step. The existence of two possible conformers E and E‘ that are in rapid equilibrium is postulated, to reconcile reduced burst sizes with full activity of the mutant enzymes. Both forms can form the quaternary complex, but only the E form is capable of catalyzing phosphodiester bond formation. The fraction of the catalytically active E form varies from essentially 100% for the wild type to 38 and 32% for the mutants K631R and Y639F, respectively. Upon entering the elongation phase, the E form becomes the dominant form in all three enzymes, leading to comparable rates of elongation for the wild type and Y639F mutant. The rate of synthesis of long transcripts is markedly diminished for the K631R mutant due to decreased processivity.