A STUDY OF SUBUNIT INTERFACE RESIDUES OF FRUCTOSE-1,6-BISPHOSPHATASE BY SITE-DIRECTED MUTAGENESIS - EFFECTS ON AMP AND MG2+ AFFINITIES

The structural transformation of fructose-1,6-bisphosphatase upon bind ing of the allosteric regulator AMP dramatically changes the interacti ons across the C1-C4 (C2-C3) subunit interface of the enzyme. Asn9, Me t18, and Ser87 residues were modified by site-directed mutagenesis to probe the function of the interface residues in porcine liver fructose -1,6-bisphosphatase. The wild-type and mutant forms of the enzyme were purified to homogeneity and characterized by initial rate kinetics an d circular dichroism (CD) spectrometry. No discernible alterations in structure were observed among the wild-type and Asn9Asp, Met18Ile, Met 18Arg, and Ser87Ala mutant forms of the enzyme as measured by CD spect rometry. Kinetic analyses revealed 1.6- and 1.8-fold increases in k(ca t) with Met18Arg and Asn9Asp, respectively. The K-m for fructose 1,6-b isphosphate increased about 2-similar to 4-fold relative to that of th e wild-type enzyme in the four mutants. A 50-fold lower K-a value for Mg2+ compared with that of the wild-type enzyme was obtained for Met18 Ile with no alteration of the K-i for AMP. However, the replacement of Met18 with Arg caused a dramatic decrease in AMP affinity (20 000-fol d) without a change in Mg2+ affinity. Increases of 6- and 2-fold in th e K-i values for AMP were found with Asn9Asp and Ser87Ala, respectivel y. There was no difference in the cooperativity for AMP inhibition bet ween the wild-type and the mutant forms of fructose-1,6-bisphosphatase . This study demonstrates that the mutation of residues in the C1-C4 ( C2-C3) interface of fructose-1,6-bisphosphatase can significantly affe ct the affinity for Mg2+, which is presumably bound 30 Angstrom away. Moreover, the mutations alternatively reduce AMP and Mg2+ affinities, and this finding may be associated with the destabilization of the cor responding allosteric states of the enzyme. The kinetics and structura l modeling studies of the interface residues provide new insights into the conformational equilibrium of fructose-1,6-bisphosphatase.