Residues Distal from the Active Site That Alter Enzyme Function in M.HhaI DNA Cytosine Methyltransferase

Ten M.HhaI residues were replaced with alanine to probe the importance of distal protein elements to substrate/cofactor binding, methyl transfer, and product release. The substitutions, ranging from 6-20 A from the active site were evaluated by thermodynamic analysis, pre-steady and steady-state kinetics, to obtain Kd(AdoMet), Kd(DNA), kcat/Km(DNA), kcat, and kmethyltransfer values. For the wild-type M.HhaI, product release steps dominate catalytic turnover while the 4-fold faster internal microscopic constant kmethyltransfer presents an upper limit. The methyl transfer reaction has DeltaH and DeltaS values of 10.3 kcal/mol and -29.4 cal/(mol K), respectively, consistent with a compressed transition state similar to that observed in the gas phase. Although the ten mutants remained largely unperturbed in methyl transfer, long-range effects influencing substrate/cofactor binding and product release were observed. Positive enhancements were seen in Asp73Ala, which showed a 25-fold improvement in AdoMet affinity and in Val282Ala, which showed a 4-fold improvement in catalytic turnover. Based on an analysis of the positional probability within the C5-cytosine DNA methyltransferase family we propose that certain conserved distal residues may be important in mediating long-range effects.