MURINE DNA (CYTOSINE-5-)-METHYLTRANSFERASE - STEADY-STATE AND SUBSTRATE TRAPPING ANALYSES OF THE KINETIC MECHANISM

DNA (cytosine-5-)-methyltransferase is essential for viable mammalian development and has a central function in the determination and mainte nance of epigenetic methylation patterns. Steady-state and substrate t rapping studies were performed to better understand how the enzyme fun ctions. The catalytic efficiency was dependent on substrate DNA length . A 14-fold increase in K-m(DNA) was observed as the length decreased from 5000 to 100 base pairs and k(cat) decreased by a third. Steady-st ate analyses were used to identify the order of substrate addition ont o the enzyme and the order of product release. Double-reciprocal patte rns of velocity versus substrate concentration intersected far from th e origin and were nearly parallel. The kinetic mechanism does not appe ar to change when the DNA substrate is either 6250 or 100 base pairs i n length. Isotope trapping studies showed that the initial enzyme-AdoM et complex was not catalytically competent; however, the initial enzym e-poly(dI.dC-dI.dC) complex was observed to be competent: for catalysi s. Product inhibition studies also support a sequential ordered bi-bi kinetic mechanism in which DNA binds to the enzyme first, followed by S-adenosyl-L-methionine, and then the products S-adenosyl-L-homocystei ne and methylated DNA are released. The proposed mechanism is similar to the mechanism proposed for M.Hhal, a bacterial DNA (cytosine-5-)-me thyltransferase. Evidence for an enzyme-DNA-DNA ternary complex is als o presented.