Glutamic acid residue 98 is critical for catalysis in pig kidney fructose-1,6-bisphosphatase.

Site-specific mutagenesis has been used to replace Glu-98 with flutamine in pig kidney fructose-1,6-bisphosphatase (Fru-1,6-P(2)ase) in order to evaluate the role of this residue in catalysis. The combination of lower k(cat) and higher K(m) resulted in an approximately 12,000-fold reduction in the catalytic efficiency of the Glu-98-->Gln enzyme when compared to the wild-type enzyme. The affinity of the enzyme for Mg(2+) and for the allosteric inhibitor AMP was altered only slightly; however, cooperativity in the binding of both of these effectors was eliminated. In addition, AMP could not fully inhibit the Glu-98-->Gln enzyme. These data suggest a critical role for the carboxylate of Glu-98 both as a general base in the reaction, and in the mechanism of allosteric inhibition of the enzyme by AMP.