FUNCTION OF PRO-185 IN THE PROCYS OF CONSERVED MOTIF-IV IN THE ECORII[CYTOSINE-C5]-DNA METHYLTRANSFERASE

ProCys in the conserved sequence motif IV of [cytosine-C5]-DNA methylt ransferases is known to be part of the catalytic site, The Cys residue is directly involved in forming a covalent bond with the C6 of the ta rget cytosine, We have found that substitution of Pro-185 with either Ala or Ser resulted in a reduced rate of methyl group transfer by the EcoRII DNA methyltransferase. In addition, we observed an increase in the K-m for substrate S-adenosyl-L-methionine (AdoMet), but a decrease in the K-m for substrate DNA, This is reflected in minor changes in k (cat)/K-m for DNA, but in 10- to 100-fold reductions in k(cat)/K-m for AdoMet, This suggests that Pro-185 is important to properly orient th e activated cytosine and AdoMet for methyl group transfer by direct in teraction with AdoMet and indirectly via the Cys interaction with cyto sine.