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J. Biol. Chem., Vol. 276, Issue 24, 20924-20934, June 15, 2001

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The Mechanism of DNA Cytosine-5 Methylation
KINETIC AND MUTATIONAL DISSECTION OF HhaI METHYLTRANSFERASE^*

Giedrius Vilkaitis, Egl Merkien, Saulius Serva, Elmar Weinhold, and Saulius Klimaauskas^§

From the Institute of Biotechnology, Laboratory of Biological DNA Modification, LT-2028 Vilnius, Lithuania and  Institut für Organische Chemie der RWTH Aachen, D-52056 Aachen, Germany

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 s¹, K_D = 6 µM) is partially converted into products during isotope-partitioning experiments, suggesting that it is catalytically competent. Chemical formation of the product M.HhaI·^MeDNA·AdoHcy (k_chem = 0.26 s¹) is followed by a slower decay step (k_off = 0.045 s¹), which is the rate-limiting step in the catalytic cycle (k_cat = 0.04 s¹). 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, k, K values but small changes in K, K, 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 = 0.7 × 10³ s¹), 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.

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