title: Residues distal from the active site that alter enzyme function in M.HhaI DNA cytosine methyltransferase.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA.
authors.forename: Vyas
authors.initials: V
authors.lastname: Sharma
authors.forename: Ben
authors.initials: B
authors.lastname: Youngblood
authors.forename: Norbert
authors.initials: N
authors.lastname: Reich
mesh_terms: D020134:Catalytic Domain; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D017434:Protein Structure, Tertiary; D012436:S-Adenosylmethionine / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D012436:S-Adenosylmethionine; D004248:DNA (Cytosine-5-)-Methyltransferases