An Unusually Low pK_afor Cys282 in the Active Site of Human Muscle Creatine Kinase

Abstract

All phosphagen kinases contain a conserved cysteine residue which has been shown by crystallographic 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 have used site-directed mutagenesis to replace Cys282 of human muscle creatine kinase with serine and methionine. 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 greatly reduced, activity in both the forward (creatine phosphorylation) and reverse (MgADP phosphorylation) reactions. The K(m) for creatine was increased approximately 10-fold, but the K(m) for phosphocreatine was relatively unaffected. The V and V/K pH-profiles for the wild-type enzyme were similar to those reported for rabbit muscle creatine kinase, the most widely studied creatine kinase isozyme. However, the V/K(creatine) profile for the C282S mutant was missing a pK of 5.4. This suggests that Cys282 exists as the thiolate anion, and is necessary for the optimal binding of creatine. The low pK of Cys282 was also determined spectrophotometrically and found to be 5.6 +/- 0.1. The S284A mutant was found to have reduced catalytic activity, as well as a 15-fold increase in K(m) for creatine. The pK(a) of Cys282 in this mutant was found to be 6.7 +/- 0.1, indicating that H-bonding to Ser284 is an important, but not the sole, factor contributing to the unusually low pK(a) of Cys282.

Exploring the Role of the Active Site Cysteine in Human Muscle Creatine Kinase

“…These data, when fitted to the Henderson-Hasselbalch equation (eq 5), provided a pK a for Cys283 (in the wild-type enzyme) of 5.7 ± 0.1. This is consistent with the value of 5.6 ± 0.1 determined in a previous study (19) and demonstrates that the method is reproducible between enzyme preparations. The Δε 240 for the P284A and C283S, as well as the C283S/S285C and C283S, variants was determined over the same pH range (Figure 3).…”

“…Using a structural model involving only Cys283, Pro284 and Ser285 ( Figure 2B) and based on the coordinates of human ubiquitous mitochondrial CK (30), Naor and Jensen (25) predicted a pK a value of 6.1 for Cys283. This was in reasonable agreement with the experimental value of 5.6 (19). Removal of the Ser side-chain, thereby creating a model for the S285A variant, increased the calculated pK a value to 7.2, also in good agreement with the experimental value of 6.7 obtained for this mutant (19).…”

Loop Movement and Catalysis in Creatine Kinase

“…The H66A variant had pKs of 5.4 + 0.1 and 6.1 + 0.1 while the D326N variant had pKs of 5.6 + 0.1 and 6.1 + 0.1. These were not dissimilar to the pKs of 5.4 and 6.7, and 5.6 and 7.4, observed for WT HMCK (19) and rabbit muscle CK (21), respectively.…”

“…It appears that they accomplish this by having a sufficiently strong interaction to align, if not clamp, the two mobile loops over the active site. In doing so they orient Ile69 and Val325 to create a binding pocket for the methyl group of creatine thereby imparting a level of substrate discrimination (16) and, in concert with Glu232 and Cys282 (18,19), evidently provide the precise alignment of substrates necessary for catalysis.…”

“…The pH-rate profiles for reaction in the forward direction were carried out at a saturating concentration of MgATP (5 mM) essentially as described (19). Data for the creatine k cat / K m profile, which decreased with a final slope of 2 as the pH was decreased, were fitted to:…”