Alkaline phosphatases of Neurospora crassa. Part II product inhibition studies

Abstract

A partially purified preparation of the constitutive alkaline phosphatase from Neurospora crassa, containing two electrophoretically distinct activities was used in initial studies of product inhibition patterns. Inorganic phosphate was shown to be a linear competitive inhibitor, and p-nitrophenol to be a non-linear, non-competitive inhibitor of p-nitrophenyl phosphate hydrolysis. Glycerol was shown to be a linear non-competitive inhibitor of β-glycerophosphate hydrolysis.A purification procedure whereby one enzyme activity could be obtained free of the second was devised. The purified enzyme catalyzed the hydrolysis of a wide range of substrates and had a molecular weight of 111 000. Its hydrolysis of glucose 6-phosphate was competitively inhibited by phosphate and non-competitively inhibited by glucose. Both inhibitions were linear. Hydrolysis of p-nitrophenyl phosphate was competitively inhibited by phosphate in a linear manner, but p-nitrophenol was a non-linear, non-competitive inhibitor. Alternate product inhibition by glucose was linear competitive. No inhibition by p-nitrophenol of glucose 6-phosphate hydrolysis could be detected.The inhibition data for glucose 6-phosphate and β-glycerophosphate may be consistent with an ordered Uni-Bi mechanism expanded to include one or more isomerizations of enzyme complexes. The postulation of a different mechanism involving alternate pathways is probably required to explain the data obtained when p-nitrophenyl phosphate was the substrate.