PURIFICATION AND PROPERTIES OF A MONOFUNCTIONAL IMIDAZOLEGLYCEROL-PHOSPHATE DEHYDRATASE FROM WHEAT

Imidazoleglycerol-phosphate dehydratase (EC 4.2.1.19) activity was det ected in extracts of several monocotyledonous and dicotyledonous plant s using a newly developed assay method. The enzyme was purified 114,00 0-fold (to apparent homogeneity) from wheat germ by five chromatograph ic steps. Its native relative molecular weight (M(r)) was determined t o be 600,000 to 670,000, and it consists of identical subunits of M(r) 25,500. In wheat germ, the dehydratase, unlike those of prokaryotic o rigin, is not associated with histidinol phosphatase activity. The rea ction product was identified as imidazoleacetol phosphate (IAP) by com paring it with synthetic IAP as an authentic reference. The K-m value for imidazoleglycerol phosphate was 0.36 mM at the optimal pH of 6.6. The enzyme required a reducing agent, such as 2-mercaptoethanol or dit hiothreitol, and Mn2+ for maximal activity. 3-Amino-1,2,4-triazole com petitively inhibited the activity with a K-i value of 46 mu M. The pur ification of imidazoleglycerol-phosphate dehydratase from wheat germ a nd histidinol dehydrogenase from cabbage (A. Nagai, A. Scheidegger [19 91] Arch Biochem Biophys 284: 127-132) suggests that at least the seco nd half of the histidine biosynthesis in plants is identical to that i n microorganisms.