Reaction of brain hexokinase with tetranitromethane: oxidation of essential thiol groups

Subbarao, B. ; Kenkare, Umakant W. (1977) Reaction of brain hexokinase with tetranitromethane: oxidation of essential thiol groups Archives of Biochemistry and Biophysics, 181 (1). pp. 8-18. ISSN 0003-9861

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Treatment of bovine brain mitochondrial hexokinase with a fivefold molar excess of tetranitromethane (TNM) at pH 8.0 results in a complete loss of activity of the enzyme. Spectral measurements and amino acid analysis showed that only cysteinyl residues were modified by TNM under these conditions. The products of the reaction of TNM with the enzyme were cysteic acid and disulfides as found by amino acid analysis of the carboxymethylated hexokinase. Failure of 2-mercaptoethanol to reverse the effect of TNM suggested that the thiol group that is oxidized to cysteic acid is the one essential for enzyme activity. Substrates glucose and ATP, inhibitors ADP and glucose 6-phosphate, and the effector Pi partially protect against the inactivation of the enzyme by TNM. Studies with the carboxymethylated and performic acid-oxidized enzyme showed that it contained disulfide bonds when stored in the absence of 2-mercaptoethanol. The total number of half-cystines in hexokinase, present as cysteines and disulfides, is estimated to be about 20. A study of the reaction of TNM with the inactive enzyme derivative, prepared by reaction with stoichiometric amounts of 5,5'-dithiobis(2-nitrobenzoic acid) [Redkar, V. D., and Kenkare, U. W. (1975), Biochemistry, 14, 4704], indicated that one more thiol, thus far not implicated in enzyme function, is important for the active conformation of the enzyme.

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