MUTATION OF ARGININE-276 TO METHIONINE CHANGES MG2+ COOPERATIVITY AND THE KINETIC MECHANISM OF FRUCTOSE-1,6-BISPHOSPHATASE

Arginine 276 of porcine liver fructose-1,6-bisphosphatase (FBPase) was mutated to methionine by site-directed mutagenesis on the basis of the crystal structure of the enzyme [Zhang, Y., Liang, J,-Y., Huang, S., Ke, H., & Lipscomb, W. N. (1993) Biochemistry 32, 1844-1857]. The mutant and wild-type forms of the enzyme were purified to homogeneity and characterized by circular dichroism spectrometry (CD) and initial-rate kinetics, There were no discernible differences between the secondary structures of the wild-type and the mutant enzymes on the basis of the CD data. Replacement of Arg 276 with methionine caused a significant decrease in the enzyme's activity. The k(cat) for the mutant enzyme was only about 0.67% of that of the wild-type enzyme. Most importantly, the mutation caused the total loss of cooperativity for Mg2+ and changed the kinetic mechanism to one in which the substrate adds to FBPase before Mg2+ and in which all steps equilibrate rapidly relative to the conversion of the ternary complex of enzyme, substrate, and Mg2+ to products. The K-a for Mg2+ increased by only about 5-fold relative to that of the wild-type enzyme. The mutation did not change the K-i for AMP or the Hill coefficient of this allosteric inhibitor. The K-i for fructose 2,6-bisphosphate was increased by 16-fold compared with that of the wild-type enzyme. The K-m for fructose 1,6-bisphosphate was similar to that of the wild-type enzyme. It is concluded that Arg 276 is critical for activity and Mg2+ cooperativity with FBPase and it determines the enzyme's kinetic mechanism.