L-Histidine Ammonia-lyase in Rat Liver

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Abstract
Histidine ammonia-lyase [EC 4.3.1.3] was purified approximately 390 fold from female rat liver. The enzyme preparation was shown to be homogeneous and its s20, w value was 11.6 by ultracentrifugal analysis. The molecular weight was determined to be 190,000 by the sedimentation equilibrium method. On disc-electrophoresis, purified enzyme separated into one major and two minor components having enzyme activity, but after pretreatment with reduced glutathione, the preparation showed only one band. This suggests that the enzyme preparation was pure and that a thiol group is involved in the conversion of polymeric forms to the monomeric form. The isoelectric point was determined to be pH 5.2 by pH gradient electrophoresis. On immunoelectrophoresis, the purified enzyme gave a single band against anti-histidine ammonia-lyase serum. The enzyme activity was highest between pH 8.8 and 9.0. The Km value for histidine was calculated to be 1.2xlO−3sM. Thiol compounds, such as glutathione and mercaptoethanol, considerably activated the enzyme. Parachloromercuribenzoate completely inhibited histidine ammonialyase activity at a concentration, of 10−5 M and this inhibition was partially reversed by addition of the substrate, histidine. The results indicate that a thiol is involved, not only in the conversion of the associated forms to the dissociated form, but also in the increase in catalytic activity of the enzyme. EDTA markedly inhibited enzyme activity and this inhibition was reversed effectively by Mn2+, Mg2, Zn2+, and Cd2+ and less effectively by Ca2+ and Ni2+. The activating effect of glutathione was lost in the presence of EDTA and reversed by addition of Mn2+. These results suggest that a divalent metal ion is essential for the catalytic activity of histidine ammonia-lyase and that thiol is intimately related with its function. Sodium borohydride and nitromethane, which are known to inhibit bacterial histidine ammonialyase activity by reacting with dehydroalanine in the active center, considerably inhibited the enzyme from rat liver.

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