Kinetic and Catalytic Mechanism of HhaI Methyltransferase*

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Vol. 262, No. 10,Issue of April 5 , pp. 47764786 1987 Printed in t h A .

Kinetic and Catalytic Mechanism of HhaI Methyltransferase* (Received for publication, May 12,1986)

John C. Wu and Daniel V. Santi From the Department of Biochemistry and Biophysics and the Department of Pharmaceutical Chemistry, Universityof . . California, Sun Francisco, California 94143

Kinetic and catalytic properties of the DNA (cyto- and AdoHcy. In mammalian cells, DNA (cytosine-5)-methsine-5)-methyltransferase HhaI are described. With yltransferases methylate certain CpG sequences which are poly(dG-dC) as substrate, the reactionproceeds by an believed to modulate gene expression and cell differentiation equilibrium (or processive) ordered Bi-Bi mechanism (for review see Ref. 1).Bacterial DNA (cytosine-5)-methylin which DNA binds to the enzyme first, followed by transferases are acomponent of restriction-modification sysS-adenosylmethionine (AdoMet). After methyl trans- tems and serve as valuable tools for the manipulation of DNA fer, S-adenosylhomocysteine (AdoHcy) dissociates fol- structure andthe analysis of protein-nucleic acid interactions. lowed by methylated DNA. AdoHcy is a potent comWe have undertaken studies to elucidate the mechanisms petitive inhibitor withrespect to AdoMet (Ki= 2.0 ELM) of catalysis, ligand interactions, and specificity of DNA (cyand its generation during reactions results in nonlinear kinetics. AdoMet and AdoHcy significantly in- tosine-5)-methyltransferases.Recently we proposed that the teract with only the substrate enzyme-DNA complex; catalytic mechanism of DNA (cytosine-5)-methyltransferases they do notbind to freeenzyme and bind poorly to the involves formation of a transientcovalent adduct between the methylated enzyme-DNA complex. In the absence of enzyme and the 6-carbon of cytosines, in analogy to other AdoMet, HhaI methylase catalyzes exchangeof the 5- enzymes which catalyze 1-carbon transfer to pyrimidines (2, H of substrate cytosines for protons of water atabout 3). This hypothesis is supported by observations that DNA of DNA (cytosine-5)7-fold the rate of methylation. The 5-H exchange re- containing azaC, apotentinhibitor action is inhibited by AdoMet or AdoHcy. In the en- methyltransferases, forms covalent complexes with mammazyme-DNA-AdoHcy complex, AdoHcy also suppresses lian and bacterial enzymes (4-7). The azaC is presumed to dissociation of DNA and reassociation of the enzyme form astable adduct analogous to the proposed catalytic with other substrate sequences. Our studies reveal that intermediate of the reaction. the catalytic mechanism of DNA (cytosine-5)-methylIn this paper, we describe the kinetic and catalytic mechatransferases involves attack of the C6 of substrate nisms of the DNA (cytosine-5)-methyltransferase HhaI methcytosines by an enzyme nucleophile and formationof a ylase, which recognizes the sequence GCGC. Using poly(dGtransient covalent adduct. Based on precedents of other dC), a synthetic substrate of the enzyme (8), we show that enzymes which catalyze similar reactions and the sus- the methylation reaction proceeds by an ordered kinetic ceptibility of HhaI to inactivation by N-ethylmaleim- scheme in which enzyme first binds to DNA. We also demide, we propose that thesulfhydryl groupof a cysteine onstratea novel enzyme-induced exchange of the 5-H of residue is the nucleophilic catalyst. Furthermore, we cytosines in the absence of AdoMet and provide evidence for propose that Cys-81 is the active-site catalyst in HhaI. the formation of a transient covalent intermediate during This residue is found in a Pro-Cys doublet which is methylation by HhaI methylase. conserved in all DNA (cytosine-5)-methyltransferases whose sequences have been determined to date and is MATERIALS AND METHOD$ found in related enzymes. Finally, we discuss the possibility that covalent adducts between C6 of pyrimiRESULTS dines and nucleophiles of proteins may be important Properties of HhaI Methylase-HhaI methylase is a mongeneral components of protein-nucleic acid interacomer of M, = 37,000 as determined by gel filtration chromations. tography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is rapidly inactivated by 0.4 mM NEM (tLh < 10 s at 22 “C) and is protected by poly(dG-dC) DNA (cytosine-5)-methyltransferases(EC 2.1.1.37) cata- (at 0.21 p ~ , 3tzh-60 s; at 1.1 p~ tIh -120 sf. In contrast, lyze the AdoMetl-dependent methylation of cytosine residues *The “Materials and Methods” and Fig.1-7 are presented in in specific sequences in DNA to give DNA-[5-methyl]cytosine miniprint at the endof this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, BeGrant CA14394 from the National Cancer Institute. The companion thesda, MD 20814. Request Document No. 86M-1588, cite the aupaper (14) may also be consulted. The costs of publication of this thors, and include a check or money order for $2.70 per set of article were defrayed in part by the payment of page charges. This photocopies. Full size photocopies are also included in the microfilm article must therefore be hereby marked “aduertisemnt” in accord- edition of the Journal that is available from Waverly Press. To facilitate comparison between kinetic data for HhaI methylase ance with 18 U.S.C. Section 1734 solelyto indicate this fact. concentraThe abbreviations used are: AdoMet, S-adenosyl-L-methionine; and those for other DNA (cytosine-5)-methyltransferases, AdoHcy, S-adenosyl-L-homocysteine;azaC, azacytosine; TES, N - tions of poly(dG-dC) are expressed in units of double-stranded rectris[hydroxymethyl]methyl-2-aminoethanesulfonicacid; NEM, N - ognition sites. Hence, poly(dG-dC) at 1.0 hi in total nucleotides is ethyl maleimide; HhaI methylase, HhaI DNA (cytosine-5)-methyl- 0.25 PM,or one-fourth of the nucleotide concentration, in recognition transferase from H. haemolyticrcs; HPLC, high performance liquid sites. This convention is followed throughout the text unless otherwise stated. chromatography; DTT, dithiothreitol; BSA, bovine serum albumin.

* This work was supported by United States Public Health Service

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Mechanism of HhaI Methylose AdoMet up to 25 p~ (1500 X K,,,) does not protectthe enzyme from inactivation by NEM. These data suggest that anactive site sulfhydryl group may be involved in catalysis by HhaI methylase. One picomole of enzyme is equal to 1.3 units of activity based on estimates of proteinconcentrationina purified preparation and from inhibition of 5-3H exchange by AdoHcy using the method of Henderson (38). Kinetics of HhaI Methylase-catalyzed Methylation of Poly(dG-de)-Early in this study we observed that the rate of HhaI-catalyzed methylation of poly(dG-dC) by AdoMet progressively decreased as the reaction proceeded (Fig. la). The decrease in ratewas inversely related to theconcentration of AdoMet and most noticeable at low concentrations of AdoMet (