Essential Histidyl Residues of Octopine Dehydrogenase

Octopine dehydrogenase from muscles of Pecten maximus has been treated by diethylpyrocarbonate at pH 6.0. The enzyme was completely inhibited when 2 histidyl residues were carbethoxylated. In the same conditions rabbit muscle lactate dehydrogenase looses its activity when 12 histidines are modified. The carbethoxylation of both enzymes does not follow a simple first order kinetics. The specificity of the reaction of diethylpyrocarbonate with histidyl residues has been confirmed by the reversal of the inhibition by hydroxylamine, by the lack of reaction with tyrosines and cysteines and by the absence of important structural modifications. The carbethoxylated enzyme gave a normal binary complex with the coenzyme, but the ternary complex formation with substrate analogues is disturbed. The coenzymes or the substrates used separately do not protect the enzyme against diethylpyrocarbonate. In the ternary complexes the reaction with the inhibitor is hindered. The study of the pH effect on the Km of octopine has been made in H2O and 2H2O. One of the downward bends of the curve indicates a group in the free enzyme with a pK of 6.1, which is shifted to 6.6 in 2H2O medium. This confirms the involvement of histidyl residues in the reaction catalysed by octopine dehydrogenase. The number of essential histidines in octopine dehydrogenase is discussed.