The functional role of histidine and sulfhydryl groups in rat liver thiamine pyrophosphokinase

Abstract

The functional role of histidine and sulfhydryl groups of thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, E.C.2.7.6.2) has been studied by the methods of photooxidation and chemical modification by diethylpyrocarbonate (DEPC) and Ellman's reagent, 5,5' - dithiobis (2 - nitrobenzoic acid) (DTNB). Histidine amino acid residues have been shown to be destroyed during photoinactivation. Titration of the protein with DEPC has established that modification of two histidine groups decreases the catalytic activity of thiamine pyrophosphokinase. Interaction of the subsequent three groups with the reagent does not affect residual activity. Substrates protect thiamine pyrophosphokinase from inactivation. Two -SH groups have been identified in a molecule of thiamine pyrophosphokinase with Ellman's reagent. Modification of only one of them results in complete loss of the enzymatic activity. Treatment of the enzyme with 8M urea has shown no differences in the amount of thiol groups of the native and denatured enzymes. Mg2+ + ATP and somewhat more weakly Mg2+ partially protect thiamine pyrophosphokinase from inhibition by DTNB. In their presence a high degree of enzyme reactivation is observed. Both histidine and sulfhydryl groups are suggested to play an important role in the catalytic mechanism of thiamine pyrophosphokinase.