Kinetic mechanism of cytosine DNA ethyltransferase MspI

Citation
Sk. Bhattacharya et Ak. Dubey, Kinetic mechanism of cytosine DNA ethyltransferase MspI, J BIOL CHEM, 274(21), 1999, pp. 14743-14749
Citations number
30
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
0021-9258 → ACNP
Volume
274
Issue
21
Year of publication
1999
Pages
14743 - 14749
Database
ISI
SICI code
0021-9258(19990521)274:21<14743:KMOCDE>2.0.ZU;2-O
Abstract
A kinetic analysis of MspI DNA methyltransferase (M.MspI) is presented. The enzyme catalyzes methylation of A-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 10(8) M-1 s(-1). But the values of the specificity constants fo r the smaller DNA substrates (121 and 1459 base pairs (bp)) with single met hylation target or with multiple targets (sonicated A-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 noncompet itive with respect to S-adenosylmethionine (AdoMet). The S-adenosylhomocyst eine inhibition of the methylation reaction is competitive with respect to AdoMet and uncompetitive with respect to DNA. The presteady state kinetic a nalysis showed a burst of product formation when AdoMet was added to the en zyme preincubated with the substrate DNA. The burst is followed by a consta nt rate of product formation (0.06 mol per mol of enzyme s(-1)) which is si milar to catalytic constants (k(cat) = similar to 0.056 s(-1)) measured und er steady state conditions. The isotope exchange in chasing the labeled met hyltransferase-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 c onsistent with an ordered Bi Bi mechanism for the M.MspI-catalyzed DNA meth ylation where DNA binds first.