An unusually low pK(a) for Cys282 in the active site of human muscle creatine kinase.
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.
Wang
P F
PF
College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065, USA.
McLeish
M J
MJ
Kneen
M M
MM
Lee
G
G
Kenyon
G L
GL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003402:Creatine Kinase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet
D017931:DNA Primers; D011994:Recombinant Proteins; D003402:Creatine Kinase; D003545:Cysteine