The nature and reactivity of the ‘essential’ thiol in rabbit muscle creatine kinase III (EC 2.7.3.2)

• Published in: Biochemical and biophysical research communications Vol. 107; no. 1; pp. 302 - 306
• Main Authors: Fawcett, Alison H., Keto, Aila I., Mackerras, Paul, Hamilton, Susan E., Zerner, Burt
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.07.1982
• Subjects: 2-chloromercuri-4-nitrophenol; Animals; Binding Sites; chemical modification; Chloromercurinitrophenols - pharmacology; creatine kinase; Creatine Kinase - metabolism; Isoenzymes; Kinetics; Macromolecular Substances; muscles; Muscles - enzymology; Phenylmercury Compounds - pharmacology; Protein Binding; Rabbits; Sulfhydryl Reagents - pharmacology
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/0006-291X(82)91704-1

Rabbit muscle creatine kinase III (EC 2.7.3.2) can be reacted with 2-chloromercuri-4-nitrophenol and this results in the incorporation of two moles of mercurial per mole of enzyme subunit in a biphasic reaction. The second-order rate constant for the slow reaction is 475 ± 42 M −1 s −1. S-Carboxamidomethyl-creatine kinase reacts with a single mole of mercurial per mole of subunit. The rate constant, 466 ± 57 M −1 s −1, is almost identical to that for the slow reaction of the native enzyme. The reaction between 3-carboxy-4-nitrophenylthio-creatine kinase and 2-chloromercuri-4-nitrophenol has a second-order rate constant of 449 ± 56 M −1 s −1. The results may be explained if the mercurial reacts very rapidly with that cysteine residue which reacts independently with iodoacetamide or 5,5′-dithiobis(2-nitrobenzoic acid). However, 2-chloromercuri-4-nitrophenol also reacts more slowly with a second cysteine residue. Definition of the essentiality of thiol groups in enzymes by reaction with labile ligands, here represented by organomercurials, clearly must be approached with caution.