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Analysis of the role of His232 in the mechanism Ps. putida creatinase
Author(s) -
Gustely Erin E.,
Snider Mark J.
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.a43-d
Subject(s) - biochemistry , histidine , enzyme , mutagenesis , mutant , site directed mutagenesis , recombinant dna , biology , gene , plasmid , microbiology and biotechnology , chemistry
Creatine amidinohydrolase (creatinase) is an important enzyme in creatine metabolism. Catalyzing the hydrolysis of creatine to sarcosine and urea, this enzyme has been characterized in several bacteria and may also be found in humans. Increased creatinase activity has been linked to Duchenne Muscular Dystrophy, a genetic, degenerative disease. Mechanistic studies of creatinase have been limited to structural analyses that suggest the involvement of a histidine as a general base catalyst, yet the role of this histidine has not yet been functionally characterized. To this end, this research involves cloning the creatinase gene from Ps. putida into the pMAL‐c2G plasmid, creating a His232A enzyme variant via site‐directed mutagenesis, purifying the wild‐type and mutant creatinase enzymes, and comparing their kinetic mechanisms through a spectrophotometric based coupled‐assay. Currently, the creatinase gene has been cloned, as verified by DNA sequencing analysis, and transformed into the BL21(DE3)pLysS competent cell line for protein expression. Results of mutagenesis, purification, and subsequent kinetic analyses of the recombinant wild‐type and His variant enzymes will be presented.