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Abstract

1. Myokinase (ATP-AMP-phosphotransferase, EC 2.7.4.3) contains no methylated histidine residues. 2. 2. The reagent 5-diazonium-1-H-tetrazole reacted preferentially with the thiol groups of myokinase. When the SH groups were protected against azotation, the tyrosine and lysine groups reacted before histidine. As far as specific color reactions are concerned, like in other proteins ideal “bisazohistidine” plateaus were found but they were largely due to the absorption of monoazotyrosine. 3. 3. Myokinase could be reversibly inactivated by monodansylation. In monodansylmyokinase one histidine residue was protected against photochemical oxidation and against azotation. This result must be interpreted with care because dansylation leads to dimerization of myokinase. 4. 4. From photochemical oxidation experiments it follows that in myokinase there is no correlation between the loss of activity and the oxidation of the “fast” histidine. Maximally one of two “slow” histidines can be essential for enzyme activity. 5. 5. The results of photochemical oxidation confirm that two methionine residues may be involved in substrate binding and/or catalysis. 6. 6. A “destruction-reduction method” for the determination of methionine sulfoxide in proteins is introduced. It is based on the selective destruction of methionine by CNBr and the reduction of methionine sulfoxide by mercaptans during the acid hydrolysis of the protein.