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 inc rease in fluorescence intensity at 410 nm. The modified enzyme showed a characteristic absorption peak at 336 nm. These evidences suggested that the mechanism of inhibition of creatine kinase by o-phthalaldehyd e involves binding of the thiol and the epsilon-amino group of enzyme leading to the formation of isoindole derivative. None of the substrat es, except Mg-ATP, provided protection against o-phthalaldehyde inhibi tion. This was corroborated by fluorescence studies. Double inhibition experiments showed that p-chloromercuricphenyl sulphonic acid, a thio l-specific reagent, binds to the same cysteine which is also involved in the o-phthalaldehyde reaction. Stoichiometric results indicated tha t 2 mol of o-phthalaldehyde were incorporated per mole of enzyme molec ule upon complete inactivation. Denaturation of creatine kinase by ure a or heat treatment prior to o-phthalaldehyde addition resulted in the decrease of fluorescence intensity indicating that native conformatio n of the enzyme is essential for isoindole derivative formation.