Evidence for proximal cysteine and lysine residues present at the nucleotide domain of rabbit muscle creatine kinase.

Inactivation of rabbit muscle creatine kinase by o-phthalaldehyde was investigated. The loss of enzyme activity was concomitant with the increase in fluorescence intensity at 410 nm. The modified enzyme showed a characteristic absorption peak at 336 nm. These evidences suggested that the mechanisms of inhibition of creatine kinase by o-phthalaldehyde involves binding of the thiol and the epsilon-amino group of enzyme leading to the formation of isoindole derivative. None of the substrates, except Mg-ATP, provided protection against o-phthalaldehyde inhibition. This was corroborated by fluorescence studies. Double inhibition experiments showed that p-chloromercuricphenyl sulphonic acid, a thiol-specific reagent, binds to the same cysteine which is also involved in the o-phthalaldehyde reaction. Stoichiometric results indicated that 2 mol of o-phthalaldehyde were incorporated per mole of enzyme molecule upon complete inactivation. Denaturation of creatine kinase by urea or heat treatment prior to o-phthalaldehyde addition resulted in the decrease of fluorescence intensity indicating that native conformation of the enzyme is essential for isoindole derivative formation.