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J. Biol. Chem., Vol. 277, Issue 1, 279-286, January 4, 2002

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Bacteriophage T4 Dam DNA-[N⁶-adenine]Methyltransferase
KINETIC EVIDENCE FOR A CATALYTICALLY ESSENTIAL CONFORMATIONAL CHANGE IN THE TERNARY COMPLEX^*

Alexey A. Evdokimov, Victor V. Zinoviev, Ernst G. Malygin, Samuel L. Schlagman^§, and Stanley Hattman^§¶

From the  Institute of Molecular Biology, State Research Center of Virology and Biotechnology Vector, Novosibirsk 630559, Russia and the ^§ Department of Biology, University of Rochester, Rochester, New York 14627-0211

We carried out a steady state kinetic analysis of the bacteriophage T4 DNA-[N⁶-adenine]methyltransferase (T4 Dam) mediated methyl group transfer reaction from S-adenosyl-L-methionine (AdoMet) to Ade in the palindromic recognition sequence, GATC, of a 20-mer oligonucleotide duplex. Product inhibition patterns were consistent with a steady state-ordered bi-bi mechanism in which the order of substrate binding and product (methylated DNA, DNA^Me and S-adenosyl-L-homocysteine, AdoHcy) release was AdoMetDNADNA^MeAdoHcy. A strong reduction in the rate of methylation was observed at high concentrations of the substrate 20-mer DNA duplex. In contrast, increasing substrate AdoMet concentration led to stimulation in the reaction rate with no evidence of saturation. We propose the following model. Free T4 Dam (initially in conformational form E) randomly interacts with substrates AdoMet and DNA to form a ternary T4 Dam-AdoMet-DNA complex in which T4 Dam has isomerized to conformational state F, which is specifically adapted for catalysis. After the chemical step of methyl group transfer from AdoMet to DNA, product DNA^Me dissociates relatively rapidly (k_off = 1.7 s¹) from the complex. In contrast, dissociation of product AdoHcy proceeds relatively slowly (k_off = 0.018 s¹), indicating that its release is the rate-limiting step, consistent with k_cat = 0.015 s¹. After AdoHcy release, the enzyme remains in the F conformational form and is able to preferentially bind AdoMet (unlike form E, which randomly binds AdoMet and DNA), and the AdoMet-F binary complex then binds DNA to start another methylation cycle. We also propose an alternative pathway in which the release of AdoHcy is coordinated with the binding of AdoMet in a single concerted event, while T4 Dam remains in the isomerized form F. The resulting AdoMet-F binary complex then binds DNA, and another methylation reaction ensues. This route is preferred at high AdoMet concentrations.

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