Kinetic and catalytic properties of the DNA (cytosine-5)-methyltransferase HhaI 
are described. With poly(dG-dC) as substrate, the reaction proceeds by an 
equilibrium (or processive) ordered Bi-Bi mechanism in which DNA binds to the 
enzyme first, followed by S-adenosylmethionine (AdoMet). After methyl transfer, 
S-adenosylhomocysteine (AdoHcy) dissociates followed by methylated DNA. AdoHcy 
is a potent competitive inhibitor with respect to AdoMet (Ki = 2.0 microM) and 
its generation during reactions results in non-linear kinetics. AdoMet and 
AdoHcy significantly interact with only the substrate enzyme-DNA complex; they 
do not bind to free enzyme and bind poorly to the methylated enzyme-DNA complex. 
In the absence of AdoMet, HhaI methylase catalyzes exchange of the 5-H of 
substrate cytosines for protons of water at about 7-fold the rate of 
methylation. The 5-H exchange reaction is inhibited by AdoMet or AdoHcy. In the 
enzyme-DNA-AdoHcy complex, AdoHcy also suppresses dissociation of DNA and 
reassociation of the enzyme with other substrate sequences. Our studies reveal 
that the catalytic mechanism of DNA (cytosine-5)-methyltransferases involves 
attack of the C6 of substrate cytosines by an enzyme nucleophile and formation 
of a transient covalent adduct. Based on precedents of other enzymes which 
catalyze similar reactions and the susceptibility of HhaI to inactivation by 
N-ethylmaleimide, we propose that the sulfhydryl group of a cysteine residue is 
the nucleophilic catalyst. Furthermore, we propose that Cys-81 is the 
active-site catalyst in HhaI. This residue is found in a Pro-Cys doublet which 
is conserved in all DNA (cytosine-5)-methyltransferases whose sequences have 
been determined to date and is found in related enzymes. Finally, we discuss the 
possibility that covalent adducts between C6 of pyrimidines and nucleophiles of 
proteins may be important general components of protein-nucleic acid 
interactions.