STUDY OF THE ROLES OF ARG-104 AND ARG-225 IN THE 2-KINASE DOMAIN OF 6-PHOSPHOFRUCTO-2-KINASE FRUCTOSE-2,6-BISPHOPHATASE BY SITE-DIRECTED MUTAGENESIS/

The roles of Arg-104 and Arg-225 located in the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase (PFK-2)/ fructose-2,6-bi sphosphatase (FBPase-2) have been studied by site-directed mutagenesis . In recombinant rat liver PFK-2/ FBPase-2, mutation of Arg-225 to Ser increased the K-m of PFK-2 for fructose-6-phosphate (Fru-6-P) 7-fold at pH 6 and decreased PFK-2 activity at suboptimal substrate concentra tions between pH 6 and 9.5. The mutation had no effect on the V-max. o f PFK-2 or on the K-m of PFK-2 for MgATP. The mutation also increased the V-max. of FBPase-2 4-fold without changing the K, for Fru-2,6-P-2 or IC50 of Fru-6-P. These findings are in agreement with a previous st udy [Rider and Hue (1992) Eur. J. Biochem. 207, 967-972] on the protec tion by Fru-6-P of the labelling of Arg-225 by phenylglyoxal, and sugg est that Arg-225 participates in Fru-6-P binding. In recombinant rat m uscle PFK-2/ FBPase-2, mutation of Arg-104 to Ser increased the K-m fo r Fru-6-P 60-fold, increased the IC,, of citrate, increased the V-max. 1.5-3-fold at pH 8.5 and altered the pH profile of PFK-2 activity. It did not affect the K-m of PFK-2 for MgATP. The mutation also decrease d the V-max. of FBPase-2 3-fold, increased the K-m for Fru-2,6-P-2 70- fold and increased the IC50 of Fru-6-P at least 300-fold. Although the dimeric structure was maintained in the mutant, its PFK-2 activity wa s more Sensitive towards inactivation by guanidinium chloride than the wild-type enzyme activity. The findings indicate that Arg-104 is invo lved in Fru-6-P binding in the PFK-2 domain and that it might also bin d citrate. Structural changes resulting from the mutation might be res ponsible for the changes in kinetic properties of FBPase-2.