Inactivation of rabbit muscle creatine kinase by reversible formation of an internal disulfide bond induced by the fungal toxin gliotoxin.
The biological activity of gliotoxin is dependent on the presence of a strained disulfide bond that can react with accessible cysteine residues on proteins. Rabbit muscle creatine kinase contains 4 cysteines per 42-kDa subunit and is active in solution as a dimer. Only Cys-282 has been identified as essential for activity. Modification of this residue results in loss of activity of the enzyme. Treatment of creatine kinase with gliotoxin resulted in a time-dependent loss of activity abrogated in the presence of reducing agents. Activity was restored when the inactivated enzyme was treated with reducing agents. Inactivation of creatine kinase by gliotoxin was accompanied by the formation of a 37-kDa form of the enzyme. This oxidized form of creatine kinase was rapidly reconverted to the 42-kDa species by the addition of reducing agents concomitant with restoration of activity. A 1:1 mixture of the oxidized and reduced monomer forms of creatine kinase as shown on polyacrylamide gel electrophoresis was equivalent to the activity of the fully reduced form of the enzyme consistent with only one reduced monomer of the dimer necessary for complete activity. Conversion of the second monomeric species of the dimer to the oxidized form by gliotoxin correlated with loss of activity. Our data are consistent with gliotoxin inducing the formation of an internal disulfide bond in creatine kinase by initially binding and possibly activating a cysteine residue on the protein, followed by reaction with a second neighboring thiol. The recently published crystal structure of creatine kinase suggests the disulfide is formed between Cys-282 and Cys-73.
Hurne
A M
AM
Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra.
Chai
C L
CL
Waring
P
P
D000818:Animals; D003402:Creatine Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000494:pharmacology*; D005912:Gliotoxin / Q000494:pharmacology*; D005978:Glutathione / Q000494:pharmacology; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008954:Models, Biological; D009132:Muscles / Q000187:drug effects / Q000201:enzymology*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011817:Rabbits; D019163:Reducing Agents / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013997:Time Factors
D004220:Disulfides; D005656:Fungal Proteins; D007166:Immunosuppressive Agents; D019163:Reducing Agents; D013438:Sulfhydryl Compounds; D005912:Gliotoxin; D003402:Creatine Kinase; D005978:Glutathione; D003545:Cysteine; D010100:Oxygen; D004229:Dithiothreitol