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J. Biol. Chem., Vol. 279, Issue 51, 53248-53258, December 17, 2004

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Substrate Specificity and Kinetic Mechanism of Mammalian G9a Histone H3 Methyltransferase^*

Debasis Patnaik, Hang Gyeong Chin, Pierre-Olivier Estève, Jack Benner, Steven E. Jacobsen, and Sriharsa Pradhan¶

From the New England Biolabs, Beverly, Massachusetts 01915, and the Department of Molecular Cell and Developmental Biology, and Molecular Biology Institute, University of California, Los Angeles, California 90095

Lysine-specific murine histone H3 methyltransferase, G9a, was expressed and purified in a baculovirus expression system. The primary structure of the recombinant enzyme is identical to the native enzyme. Enzymatic activity was favorable at alkaline conditions (>pH 8) and low salt concentration and virtually unchanged between 25 and 42 °C. Purified G9a was used for substrate specificity and steady-state kinetic analysis with peptides representing un- or dimethylated lysine 9 histone H3 tails with native lysine 4 or with lysine 4 changed to alanine (K4AK9). In vitro methylation of the H3 tail peptide resulted in trimethylation of Lys-9 and the reaction is processive. The turnover number (k_cat) for methylation was 88 and 32 h^–1 on the wild type and K4AK9 histone H3 tail, respectively. The Michaelis constants for wild type and K4AK9 () were 0.9 and 1.0 µM and for S-adenosyl-L-methionine () were 1.8 and 0.6 µM, respectively. Comparable kinetic constants were obtained for recombinant histone H3. The conversion of K4AK9 di- to trimethyl-lysine was 7-fold slower than methyl group addition to unmethylated peptide. Preincubation studies showed that G9a-AdoMet and G9a-peptide complexes are catalytically active. Initial velocity data with peptide and S-adenosyl-L-methionine (AdoMet) and product inhibition studies with S-adenosyl-L-homocysteine were performed to assess the kinetic mechanism of the reaction. Double reciprocal plots and preincubation studies revealed S-adenosyl-L-homocysteine as a competitive inhibitor to AdoMet and mixed inhibitor to peptide. Trimethylated peptides acted as a competitive inhibitor to substrate peptide and mixed inhibitor to AdoMet suggesting a random mechanism in a Bi Bi reaction for recombinant G9a where either substrate can bind first to the enzyme, and either product can release first.

Received for publication, August 20, 2004 , and in revised form, October 12, 2004.

^* This work was supported by New England Biolabs and National Institutes of Health Grant GM60398 (to S. E. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 978-927-5054 (ext. 227); Fax: 978-921-1350; E-mail: pradhan{at}neb.com.

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