Site-directed alteration of Glu197 and Glu66 in a pyruvoyl-dependent histidine decarboxylase.

Site-directed mutagenesis has been used to explore the role of two carboxylates in the active site of histidine decarboxylase from Lactobacillus 30a. The most striking observation is that conversion of Glu197 to either Gln or Asp causes a major decrease in catalytic rate while enhancing substrate binding. This is consistent with models based on X-ray diffraction results which suggest that the acid may protonate a reaction intermediate during catalysis. The Asp197 protein undergoes a suicide reaction with substrate, apparently triggered by inappropriate protonation of the intermediate. This leads to decarboxylation-dependent transamination which converts the pyruvoyl cofactor to an alanine, inactivating the enzyme. Conversion of Glu66 to Gln affects parameters of kinetic cooperativity. The mutation fixes the Hill number at approximately 1.5, midway between the pH-dependent values of the wild-type enzyme.