Investigating the Role of Threonine-58 in Creatine Kinase Activity and Active Site Communication
Creatine kinase, a member of the phosphagen kinase family, catalyzes the reversible transfer of the ?-phosphoryl group of adenosine triphosphate (ATP) to creatine to produce adenosine diphosphate (ADP), phosphocreatine (PCr), and a proton. This reaction is important for establishing and maintaining necessary [ATP]/[ADP] concentrations in cells that have a large flux in energy including cardiac muscle, neurons, and spermatozoa. Rabbit muscle creatine kinase is an 86kDa homodimer which is a representative of muscle creatine kinases. The reason for creatine kinase's dimerization is unknown but it is hypothesized it provides a cooperative mechanism between active sites. Although creatine kinase is an asymmetrical protein, it demonstrates similar catalytic and structural features to other phosphagen kinases. One unique feature is a guanidino substrate specificity loop which is located near the active site and has been shown to assist in substrate binding and catalysis.Threonine-58 is located at the N-terminal base of the GS loop and appears to anchor the loop when it is in close contact to the dimer interface. Using site-directed mutagenesis, threonine-58 was mutated to alanine to elucidate its importance in cooperativity as well as kinetic activity. The mutant displayed a sequential mechanism where creatine bound before ATP but with half the affinity for creatine compared to wild-type. It also showed that the chemical reaction was the rate limiting step with product release influencing the reaction. T58A underwent a DTNB analysis to evaluate whether the subunits expressed half-site reactivity. The mutant was kinetically analyzed to study its overall activity as well as figure out the enzyme's binding mechanism.
© Copyright 2011 Dmytri Eck