PURIFICATION, KINETICS AND INHIBITION BY ANTIMONIALS OF RECOMBINANT PHOSPHOFRUCTOKINASE FROM SCHISTOSOMA-MANSONI

We reported before on the cloning of a cDNA encoding S. mansoni PFK. I n the present investigation we established optimal conditions for expr ession of the enzyme in insect cells with high yield. The recombinant PFK was purified to homogeneity. Kinetic properties of the pure enzyme were studied with respect to its two substrates, Fru-6-P and ATP, and were compared with properties of mammalian PFK. ATP inhibited the par asite enzyme only at concentrations higher than those which inhibited mammalian muscle PFK. Saturation curves for Fru-6-P showed typical coo perative kinetics. AMP, cAMP and Fru-2,6-bisP activated the enzyme cau sing reduced apparent K-m for Fru-6-P and an increase in maximal activ ity. Both ATP inhibition and cooperative kinetics for Fru-6-P occur at both pH 6.9 and 8.2. This is a distinct difference from the mammalian enzyme which shows these kinetic properties only at neutral or slight ly acidic pH, but not at an alkaline pH. Recombinant PFK is more sensi tive to inhibition by the trivalent antimonials, antimony potassium ta rtrate and Stibophen, than is the mammalian heart muscle enzyme. The i nhibition is at least partially antagonized by the sulfhydryl protecti ve reagent, dithiothreitol.