| Abstract
| - All phosphagen kinases contain a conserved cysteine residue which has been shown bycrystallographic studies, on both creatine kinase and arginine kinase, to be located in the active site.There are conflicting reports as to whether this cysteine is essential for catalysis. In this study we haveused site-directed mutagenesis to replace Cys282 of human muscle creatine kinase with serine andmethionine. In addition, we have replaced Cys282, conserved across all creatine kinases, with alanine.No activity was found with the C282M mutant. The C282S mutant showed significant, albeit greatlyreduced, activity in both the forward (creatine phosphorylation) and reverse (MgADP phosphorylation)reactions. The Km for creatine was increased approximately 10-fold, but the Km for phosphocreatine wasrelatively unaffected. The V and V/K pH-profiles for the wild-type enzyme were similar to those reportedfor rabbit muscle creatine kinase, the most widely studied creatine kinase isozyme. However, the V/Kcreatineprofile for the C282S mutant was missing a pK of 5.4. This suggests that Cys282 exists as the thiolateanion, and is necessary for the optimal binding of creatine. The low pK of Cys282 was also determinedspectrophotometrically and found to be 5.6 ± 0.1. The S284A mutant was found to have reduced catalyticactivity, as well as a 15-fold increase in Km for creatine. The pKa of Cys282 in this mutant was found tobe 6.7 ± 0.1, indicating that H-bonding to Ser284 is an important, but not the sole, factor contributing tothe unusually low pKa of Cys282.
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