Kinetic mechanism of cytosine DNA methyltransferase MspI

Sanjoy K. Bhattacharya, Ashok K. Dubey

Research output: Contribution to journalArticlepeer-review

46 Scopus citations

Abstract

A kinetic analysis of MspI DNA methyltransferase (M.MspI) is presented. The enzyme catalyzes methylation of λ-DNA, a 50-kilobase pair linear molecule with multiple M.MspI-specific sites, with a specificity constant (k(cat)/K(M)) of 0.9 x 108 M-1 s-1. But the values of the specificity constants for the smaller DNA substrates (121 and 1459 base pairs (bp)) with single methylation target or with multiple targets (sonicated λ-DNA) were less by an order of magnitude. Product inhibition of the M.MspI-catalyzed methylation reaction by methylated DNA is competitive with respect to DNA and noncompetitive with respect to S-adenosylmethionine (AdoMet). The S- adenosylhomocysteine inhibition of the methylation reaction is competitive with respect to AdoMet and uncompetitive with respect to DNA. The presteady state kinetic analysis showed a burst of product formation when AdoMet was added to the enzyme preincubated with the substrate DNA. The burst is followed by a constant rate of product formation (0.06 mol per tool of enzyme s-1) which is similar to catalytic constants (k(cat) = ~0.056 s-1) measured under steady state conditions. The isotope exchange in chasing the labeled methyltransferase-DNA complex with unlabeled DNA and AdoMet leads to a reduced burst as compared with the one involving chase with labeled DNA and AdoMet. The enzyme is capable of exchanging tritium at C-5 of target cytosine in the substrate DNA in the absence of cofactor AdoMet. The kinetic data are consistent with an ordered Bi Bi mechanism for the M.MspI-catalyzed DNA methylation where DNA binds first.

Original languageEnglish (US)
Pages (from-to)14743-14749
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Issue number21
DOIs
StatePublished - May 21 1999
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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