Woody A Y, Osumi-Davis P A, Hiremath M M, Woody R W
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins 80523, USA.
Biochemistry. 1998 Nov 10;37(45):15958-64. doi: 10.1021/bi9805801.
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 microM at 21 degreesC, 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.
在允许单个核苷酸添加到起始二核苷酸而不干扰酶 - DNA解离或蛋白质循环利用的条件下，对转录起始的动力学机制进行了研究。前稳态动力学研究给出了野生型及其活性位点突变体K631R和Y639F在21℃时的聚合速率常数分别为3.9、5.9和3.9 s⁻¹，反向聚合速率常数分别为3.2、2.1和2.8 s⁻¹，以及进入核苷酸的解离常数分别为26、49和24 μM。结果提示了一个模型，其中K631与进入底物的磷酸基团相互作用。结合物种的内部平衡常数接近1，这与其他磷酸转移酶的值一致。化学键形成的速率常数比产物解离的速率常数至少高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。推测存在处于快速平衡的两种可能构象E和E'，以解释突变酶的爆发大小减小但仍具有完全活性的现象。两种形式都能形成四聚体复合物，但只有E形式能够催化磷酸二酯键的形成。催化活性E形式的比例从野生型的基本上100%分别变化到突变体K631R和Y639F的38%和32%。进入延伸阶段后，E形式在所有三种酶中都成为主要形式，导致野生型和Y639F突变体的延伸速率相当。由于持续性降低，K631R突变体的长转录本合成速率显著降低。
