Vilkaitis G, Merkiene E, Serva S, Weinhold E, Klimasauskas S
Institute of Biotechnology, Laboratory of Biological DNA Modification, LT-2028 Vilnius, Lithuania.
J Biol Chem. 2001 Jun 15;276(24):20924-34. doi: 10.1074/jbc.M101429200. Epub 2001 Mar 29.
Kinetic and binding studies involving a model DNA cytosine-5-methyltransferase, M.HhaI, and a 37-mer DNA duplex containing a single hemimethylated target site were applied to characterize intermediates on the reaction pathway. Stopped-flow fluorescence studies reveal that cofactor S-adenosyl-l-methionine (AdoMet) and product S-adenosyl-l-homocysteine (AdoHcy) form similar rapidly reversible binary complexes with the enzyme in solution. The M.HhaI.AdoMet complex (k(off) = 22 s(-)1, K(D) = 6 microm) is partially converted into products during isotope-partitioning experiments, suggesting that it is catalytically competent. Chemical formation of the product M.HhaI.(Me)DNA.AdoHcy (k(chem) = 0.26 s(-)1) is followed by a slower decay step (k(off) = 0.045 s(-)1), which is the rate-limiting step in the catalytic cycle (k(cat) = 0.04 s(-)1). Analysis of reaction products shows that the hemimethylated substrate undergoes complete (>95%) conversion into fully methylated product during the initial burst phase, indicating that M.HhaI exerts high binding selectivity toward the target strand. The T250N, T250D, and T250H mutations, which introduce moderate perturbation in the catalytic site, lead to substantially increased K(D)(DNA(ternary)), k(off)(DNA(ternary)), K(M)(AdoMet(ternary)) values but small changes in K(D)(DNA(binary)), K(D)(AdoMet(binary)), k(chem), and k(cat). When the target cytosine is replaced with 5-fluorocytosine, the chemistry step leading to an irreversible covalent M.HhaI.DNA complex is inhibited 400-fold (k(chem)(5FC) = 0.7 x 10(-)3 s(-)1), and the Thr-250 mutations confer further dramatic decrease of the rate of the covalent methylation k(chem). We suggest that activation of the pyrimidine ring via covalent addition at C-6 is a major contributor to the rate of the chemistry step (k(chem)) in the case of cytosine but not 5-fluorocytosine. In contrast to previous reports, our results imply a random substrate binding order mechanism for M.HhaI.
运用动力学和结合研究，以一种模型DNA胞嘧啶-5-甲基转移酶M.HhaI和一个含有单个半甲基化靶位点的37聚体DNA双链体为研究对象，来表征反应途径中的中间体。停流荧光研究表明，辅因子S-腺苷-L-甲硫氨酸（AdoMet）和产物S-腺苷-L-高半胱氨酸（AdoHcy）在溶液中与该酶形成类似的快速可逆二元复合物。在同位素分配实验中，M.HhaI.AdoMet复合物（k(off)=22 s(-1)，K(D)=6 μM）部分转化为产物，这表明它具有催化活性。产物M.HhaI.(Me)DNA.AdoHcy的化学形成（k(chem)=0.26 s(-1)）之后是一个较慢的衰减步骤（k(off)=0.045 s(-1)），这是催化循环中的限速步骤（k(cat)=0.04 s(-1)）。对反应产物的分析表明，在初始爆发阶段，半甲基化底物完全（>95%）转化为完全甲基化产物，这表明M.HhaI对靶链具有高结合选择性。T250N、T250D和T250H突变在催化位点引入了适度扰动，导致K(D)(DNA(三元))、k(off)(DNA(三元))、K(M)(AdoMet(三元))值大幅增加，但K(D)(DNA(二元))、K(D)(AdoMet(二元))、k(chem)和k(cat)变化较小。当靶胞嘧啶被5-氟胞嘧啶取代时，导致不可逆共价M.HhaI.DNA复合物形成的化学步骤被抑制400倍（k(chem)(5FC)=0.7×10(-3) s(-1)），并且苏氨酸-250突变使共价甲基化速率k(chem)进一步显著降低。我们认为，在胞嘧啶而非5-氟胞嘧啶的情况下，通过在C-6处共价加成激活嘧啶环是化学步骤速率（k(chem)）的主要贡献因素。与先前的报道相反，我们的结果暗示M.HhaI存在随机底物结合顺序机制。
