title: Kinetic and catalytic mechanism of HhaI methyltransferase.
abstract: 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.
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D015257:DNA-Cytosine Methylases; D007700:Kinetics; D008745:Methylation; D009690:Nucleic Acid Conformation; D011089:Polydeoxyribonucleotides; D012435:S-Adenosylhomocysteine / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D014316:Tritium
chemical_list: D011089:Polydeoxyribonucleotides; D014316:Tritium; C019530:poly(dC-dG); D012436:S-Adenosylmethionine; D004247:DNA; D012435:S-Adenosylhomocysteine; C037664:DNA modification methylase HhaI; D015257:DNA-Cytosine Methylases; D004248:DNA (Cytosine-5-)-Methyltransferases