Published online 13 January 2005
Article |
Probing a rate-limiting step by mutational perturbation of AdoMet binding in the HhaI methyltransferase
Merkien
auskas*Laboratory of Biological DNA Modification, Institute of Biotechnology LT-02241 Vilnius, Lithuania
*To whom correspondence should be addressed. Tel: +370 5 260 2114; Fax: +370 5 260 2116; Email: klimasau{at}ibt.lt
Received October 14, 2004. Revised December 2, 2004. Accepted December 20, 2004.
DNA methylation plays important roles via regulation of numerous cellular mechanisms in diverse organisms, including humans. The paradigm bacterial methyltransferase (MTase) HhaI (M.HhaI) catalyzes the transfer of a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) onto the target cytosine in DNA, yielding 5-methylcytosine and S-adenosyl-L-homocysteine (AdoHcy). The turnover rate (k cat) of M.HhaI, and the other two cytosine-5 MTases examined, is limited by a step subsequent to methyl transfer; however, no such step has so far been identified. To elucidate the role of cofactor interactions during catalysis, eight mutants of Trp41, which is located in the cofactor binding pocket, were constructed and characterized. The mutants show full proficiency in DNA binding and base-flipping, and little variation is observed in the apparent methyl transfer rate k chem as determined by rapid-quench experiments using immobilized fluorescent-labeled DNA. However, the Trp41 replacements with short side chains substantially perturb cofactor binding (100-fold higher 
and 
) leading to a faster turnover of the enzyme (10-fold higher k cat). Our analysis indicates that the rate-limiting breakdown of a long-lived ternary product complex is initiated by the dissociation of AdoHcy or the opening of the catalytic loop in the enzyme.