HEXOSE PHOSPHATE-BINDING SITES OF FRUCTOSE 6-PHOSPHATE,2-KINASE-FRUCTOSE 2,6-BISPHOSPHATASE

A previous chemical modification study [Kitamura et al, (1989) J, Biol . Chem. 264, 6344-6438] has shown that N-bromoacetylethanolamine phosp hate labeled specifically Cys107 of rat liver Fru 6-P,2-kinase:Fru 2,6 -Pase and the corresponding Cys of the bovine heart enzyme, leading to inactivation of kinase activity. Since Fru 6-P provided protection ag ainst the inactivation, this region of the enzyme was thought to be a Fru 6-P binding site of the kinase enzyme. To examine this possibility , oligonucleotide-directed mutagenesis has been used to alter several residues in expressed rat testis Fru 6-P,2-kinase:Fru 2,6-Pase. The ch ange of Lys100, Lys103, and Asp112 caused at most a 2-fold increase in K-m(F6P) and a 2-3-fold increase in K-m(ATP), suggesting that these r esidues are not involved in the direct binding of Fru 6-P. However, ch ange of Arg102 to Leu and to Lys resulted in a 325x and 22x, respectiv ely, increase in K-m(F6P), and change of Arg102 to Glu resulted in nea rly complete loss of the kinase activity. Change of Cys105 to Ala or S er increased K-m(F6P) about 10x. The V-max of all these mutated enzyme s except the one that changed Arg102 to Glu (R102E) was increased 10% to 85%. The kinetic parameters of Fru 2,6-Pase were not altered by the se changes. R102E formed several polymeric forms of the enzyme, includ ing a tetramer, Both R102E and an additional derivative that substitut ed Lys for Arg102 (R102K) were slightly more susceptible to guanidine inactivation than the wild-type enzyme. These results suggest that Arg 102 is essential for binding the 6-phosphate of Fru 6-P and that Cys10 5 also may play a role at the same site.