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Studies of an Important Residue Involved in the Proton Transfer in the M. HhaI Catalytic Mechanism
Author(s) -
FaKuen Shieh,
Reich Norbert
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a41
Subject(s) - chemistry , stereochemistry , dna , cytosine , methyltransferase , ternary complex , enzyme , residue (chemistry) , methyl group , catalysis , mutant , protonation , base pair , biochemistry , methylation , organic chemistry , gene , ion , alkyl
The DNA methyltransferase M. HhaI catalyzes transfer of methyl group from SAM to the cytosine base which has been flipped out of DNA duplex into the enzyme active site. Transient protonation of the cytosine ring at the endocyclic nitrogen atom N3 mediated by Glu119 residue has been proposed to facilitates nucleophilic attack on the target base. Replacement of Glu119 into Ala result in the enzyme total loss of catalytic function, and binding affinity for the cognate DNA is decreased about 103 fold . Interestingly when the Glu119 residue is replaced by the Asp, we found the catalytic activity for this mutant protein is only decreased 10 fold in comparison to the WT M. HhaI enzyme. However, the binding affinity for the cognate DNA is decreased around 103 fold, similar to the E119A mutant. Currently, our group is trying to determine the E119A M. HhaI ternary structure and to help us to understand the total loss in enzyme function upon mutation to a shorter side chain. On the other hand, hope that the E119D M. HhaI ternary structure can support Glu119 as a proton donor, and also show why the side chain shorter by only one carbon atom causes the catalytic function to decrease around 10 fold. Moreover, because the residue Glu119 locating in a motif which is highly conserved in the DNA methyltransferases, these results provide insights into catalytic mechanisms for the entire class of enzymes.