Studies on the histidine residues in pigeonpea (Cajanus cajan L.) urease

The pH dependence of pigeonpea urease catalysis reveals the presence of two ionizable groups of pKa values of 6.2±0.1 and 8.8±0.1, respectively. When urease was treated with excess diethylpyrocarbonate (DEP) at pH 6.8, a time-dependent exponential decay of its activity was observed and the pseudo-first order rate constant was proportional to the reagent concentration. The loss of activity was accompanied by a parallel increase in absorbance at 242 nm. Titration of urease with DEP revealed the presence of 12.9±0.1 ‘accessible’ histidine groups per hexameric pigeonpea urease. Hydroxylamine did not provide significant recovery of the DEP inactivated enzyme. Spectroscopic studies, circular dichroism and fluorescence show no effect of DEP on the gross conformation of urease. Irradiation of urease with visible light in the presence of small concentrations of the basic dyes like Rose Bengal or Methylene Blue at pH 6.8, brought about a time-dependent first-order decay of enzyme activity. Urea and acetohydroxamate (AHA) protect the enzyme against the inactivation by DEP or photo-oxidation. The inactivation reaction with the DEP or Rose Bengal was found to be linearly related to the blocking of 12 essential histidine groups per hexamer for complete inactivation. Since, each protein molecule is known to possess two catalytic units per hexamer hence, we propose that urease possesses at least one essential histidine per catalytic unit. The significance of these results is discussed.