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.