Some properties of the catalytic sites of imidazoleglycerol phosphate dehydratase-histidinol phosphate phosphatase, a bifunctional enzyme from Salmonella typhimurium.

Kinetic and aggregatory properties of a partially purified preparation of D-erythro-imidazoleglycerol phosphate dehydratase-histidinol phosphate phosphatase from a derepressed mutant of Salmonella typhimurium (his012$2) have been investigated. The molecular weight in crude extracts is 300,000. Purification of the enzyme causes disaggregation, the main component keing 75,000 in molecular weight. Addition of MnCIZ causes reeggregation resulting in a molecular weiglt greater than 300,000. Aminotriazole (K, = 3.2 PM) and phosphate ion (KI = 1.3 mu) competitively inhibited only the dehydratase activity. Zinc chloride, at micromolar concentrations, inhibits only the dehydratase activity. Both histidinol (KI = 52 pm) and histidine (Kr = 10 mM) are competitive inhibitors of only the phosphatase activity. Heating at 54” results in a very rapid loss of only the phosphatase activity, TI/~ = 2 min; the dehydratase activity is stable under these conditions. ImidazoIeglyceroI phosphate is not hydrolyzed and does not inhibit the phosphatase activity. Histidinol phosphate does not affect the dehydratase activity. We conclude that the dehydratase and pkosphatase active sites are separate and distinct, although they appear to reside in a single protein. The energies of activation for both substrates were determined to be 15,100 cal per mole for the hydrolysis of histidinol phosphate and 14,700 cal per mole for the dehydration of imidazoleglycerol phosphate. The optimum pH for phosphatase activity was 6.5 to 7 and was near pH 7.5 for the dehydratase activity.