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Investigating Amino Acid Residues Involved in Active Site Communication in Creatine Kinase
Author(s) -
Karia Pritesh S.,
Edmiston Paul L
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.21.5.a276-a
Subject(s) - cooperativity , phosphocreatine , chemistry , allosteric regulation , dimer , biochemistry , cooperative binding , creatine kinase , biophysics , amino acid , active site , dtnb , binding site , creatine , stereochemistry , enzyme , biology , energy metabolism , glutathione , organic chemistry , endocrinology
Creatine kinase (CK) is responsible for catalyzing the reversible transfer of high‐energy phosphate groups from phosphocreatine to MgADP forming MgATP. These reactions play a central role in maintaining energy homeostasis and buffering cellular ATP concentrations in cells with sporadically high energy requirements such as skeletal and cardiac muscle, neurons, and spermatozoa. CK primarily exists as a 86 kDa dimer in vertebrates and a few invertebrates. One hypothesis that is used to explain dimer formation in CK is that it allows for cooperativity between the two active sites. However, a mechanism of cooperativity between the subunits is still not clear. The cooperative mechanism of CK was studied by using site‐directed mutagenesis to change conserved amino acid residues that lie between the active site and dimer interface, specifically on the N‐terminal and guanidino substrate specificity loops. H25A, D21A, R340K, R340A, and â ˆ †66–69 variants of CK were prepared, expressed and purified. Cooperativity was tested by using the reaction of C282 with 5,5’‐dithiobis 2‐nitrobenzoic acid (DTNB) in the presence and absence of substrates and the transition state analog complex. The activity of the proteins was determined in the direction of phosphocreatine formation utilizing the pH stat method. The results indicate that the guanidino specificity loop may be involved in cooperativity.