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Catalytic role of His232 in the mechanism of Pseudomonas putida creatine amidinohydrolase
Author(s) -
Agidi Senyo,
Snider Mark J.
Publication year - 2007
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.21.6.a1014
Subject(s) - pseudomonas putida , chemistry , histidine , sarcosine , biochemistry , creatine , imidazole , catalysis , mutant , titration , hydrolysis , enzyme , residue (chemistry) , stereochemistry , amino acid , organic chemistry , glycine , gene
Creatine amidinohydrolase (creatinase) catalyzes the hydrolysis of creatine to sarcosine and urea. The enzyme has been isolated in several organisms and a study has indicated the presence of creatinase in humans. Structural analysis of the enzyme suggests that conserved histidine at position 232 may act as a general acid/base catalyst. To examine the functional role of this residue, the H232A creatinase variant will be created using site‐directed mutagenensis. Analyses of the kinetic mechanisms of the mutant and wild type creatinases will be compared using an isothermal titration calorimetric method to measure the change in enthalpy that accompanies product formation. The ability to rescue partial activity of the H232A mutant using imidazole will also be examined.