Residues distal from the active site that alter enzyme function in M.HhaI DNA cytosine methyltransferase |
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Authors: | Sharma Vyas Youngblood Ben Reich Norbert |
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Institution: | Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA. |
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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. |
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