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J Biol Chem, Vol. 275, Issue 7, 4912-4919, February 18, 2000

Reconciling Structure and Function in HhaI DNA Cytosine-C-5 Methyltransferase*

William M. Lindstrom Jr.Dagger , James FlynnDagger §, and Norbert O. ReichDagger §||

From the Dagger  Department of Chemistry and Biochemistry and § Program in Biochemistry and Molecular Biology, University of California, Santa Barbara, California 93106

Pre-steady state partitioning analysis of the HhaI DNA methyltransferase directly demonstrates the catalytic competence of the enzyme·DNA complex and the lack of catalytic competence of the enzyme·S-adenosyl-L-methionine (AdoMet) complex. The enzyme·AdoMet complex does form, albeit with a 50-fold decrease in affinity compared with the ternary enzyme·AdoMet·DNA complex. These findings reconcile the distinct binding orientations previously observed within the binary enzyme·AdoMet and ternary enzyme·S-adenosyl-L-homocysteine·DNA crystal structures. The affinity of the enzyme for DNA is increased 900-fold in the presence of its cofactor, and the preference for hemimethylated DNA is increased to 12-fold over unmethylated DNA. We suggest that this preference is partially due to the energetic cost of retaining a cavity in place of the 5-methyl moiety in the ternary complex with the unmethylated DNA, as revealed by the corresponding crystal structures. The hemi- and unmethylated substrates alter the fates and lifetimes of discrete enzyme·substrate intermediates during the catalytic cycle. Hemimethylated substrates partition toward product formation versus dissociation significantly more than unmethylated substrates. The mammalian DNA cytosine-C-5 methyltransferase Dnmt1 shows an even more pronounced partitioning toward product formation.

* This work was supported by National Institutes of Health Grant GM 463333 and National Science Foundation Grant MCB-9603567 (to N. O. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: EpiGenX Pharmaceuticals, 2124 Bath St., Santa Barbara, CA 93105.

|| To whom correspondence should be addressed. Tel.: 805-893-8368; Fax: 805-893-4120; E-mail: reich@chem.ucsb.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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