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Changing the Substrate Specificity of Creatine Kinase to Glycocyamine Through Mutagenesis of Non‐Conserved Residues
Author(s) -
Azevedo Ana Clara,
Fraga Dean,
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.21.6.a1010-b
Subject(s) - mutagenesis , biochemistry , kinase , mutant , creatine kinase , enzyme , site directed mutagenesis , mutation , phosphorylation , chemistry , substrate (aquarium) , biology , gene , ecology
Phosphagen kinases (PK) are a class of enzymes that catalyze the reversible phosphorylation of a guanidino substrate by MgATP. Creatine kinase (CK) and glycocyamine kinase (GK) both belong to the PK family and utilize very similar substrates. This study investigates the possible mechanisms of molecular evolution in the PK enzyme family by determining if non‐conserved residues provide a buffer to changes in functionally important residues for substrate specificity in CK and GK. Such an evolutionary mechanism theorizes that neutral mutations become randomly incorporated over time and yield a modified protein structure that would help compensate for “catastrophic” mutations which are necessary for new function. This mechanism was tested by random mutagenesis of four non‐conserved residues peripheral to the guanidino binding site in the V324E variant of CK. The V324E substitution is necessary for glycocyamine recognition, but this change alone leads to a >1000‐fold reduction in CK activity without a gain in GK activity. The mutations that recovered activity were also tested in wt CK to measure their affect on native activity. A rapid phosphomolybdate colorimetric assay was used to test the mutants for CK and GK activity. The high‐throughput screening was facilitated by using a newly developed secretion tag (YebF) and rapid affinity purification. This research was supported by NSF grant #0344432.