MODIFICATION OF CYS-128 OF PIG-KIDNEY FRUCTOSE 1,6-BISPHOSPHATASE WITH DIFFERENT THIOL REAGENTS - SIZE-DEPENDENT EFFECT ON THE SUBSTRATE AND FRUCTOSE-2,6-BISPHOSPHATE INTERACTION

Treatment of fructose 1,6-bisphosphatase with N-ethylmaleimide was sho wn to abolish the inhibition by fructose 2,6-bisphosphate, which also protected the enzyme against this chemical modification [Reyes, A., Bu rgos, M. E., Hubert, E., and Slebe, J. C. (1987), J. Biol. Chem. 262, 8451 84541. On the basis of these results, it was suggested that a sin gle reactive sulfhydryl group was essential for the inhibition. We hav e isolated a peptide bearing the N-ethylmaleimide target site and the modified residue has been identified as cysteine-128. We have further examined the reactivity of this group and demonstrated that when reage nts with bulky groups are used to modify the protein at the reactive s ulfhydryl [e.g., N-ethylmaleimide or 5,5'-dithiobis-(2-nitrobenzoate)] , most of the fructose 2,6-bisphosphate inhibition potential is lost. However, there is only partial or no loss of inhibition when smaller g roups (e.g., cyanate or cyanide) are introduced. Kinetic and ultraviol et difference spectroscopy-binding studies show that the treatment of fructose 1,6-bisphosphatase with N-ethylmaleimide causes a considerabl e reduction in the affinity of the enzyme for fructose 2,6-bisphosphat e while affinity for fructose 1,6-bisphosphate does not change. We can conclude that modification of this reactive sulfhydryl affects the en zyme sensitivity to fructose 2,6-bisphosphate inhibition by sterically interfering with the binding of this sugar bisphosphate, although thi s residue does not seem to be essential for the inhibition to occur. T he results also suggest that fructose 1,6-bisphosphate and fructose 2, 6-bisphosphate may interact with the enzyme in a different way.