DIRECTED MUTATIONS IN THE POORLY DEFINED REGION OF PORCINE LIVER FRUCTOSE 1,6-BISPBOSPHATASE SIGNIFICANTLY AFFECT CATALYSIS AND THE MECHANISM OF AMP INHIBITION

Asn(64), Asp(68), Lys(71), Lys(72), and Asp(74) of porcine liver fruct ose-1,6-bisphosphatase (FBPase) are conserved residues and part of a l oop for which no electron density has been observed in crystal structu res. Yet mutations of the above dramatically affect catalytic rates an d/or AMP inhibition. The Asp(74) --> Ala and Asp(74) --> Asn mutant en zymes exhibited 50,000- and 2,000-fold reductions, respectively, in k( cat) relative to wild-type FBPase. The pH optimum for the catalytic ac tivity of the Asp(74), Glu, Asp(68) --> Glu, Asn(64) --> Gln, and Asn( 64) --> Ala mutant enzymes shifted from pH 7.0 (wild-type enzyme) to p H 8.5, whereas the Lys(71) --> Ala mutant and Lys(71,72) --> Met doubl e mutant had optimum activity at pH 7.5. Mg2+ cooperativity, K-m for f ructose 1,6-bisphosphate, and K-i for fructose 2,6-bisphosphate were c omparable for the mutant and wild-type enzymes. Nevertheless, for the Asp(74) --> Glu, Asp(68) --> Glu, Asn(64) --> Gln, and Asn(64) --> Ala mutants, the binding affinity for Mg2+ decreased by 40-125-fold relat ive to the wild type enzyme. In addition, the Asp(74) --> Glu and Asn( 64) --> Ala mutants exhibited no AMP cooperativity, and the kinetic me chanism of AMP inhibition with respect to Mg2+ was changed from compet itive to noncompetitive. The double mutation Lys(71,72) --> Met increa sed K-i for AMP by 175-fold and increased Mg2+ affinity by 2-fold rela tive to wild-type FBPase. The results reported here strongly suggest t hat loop 51-72 is important for catalytic activity and the mechanism o f allosteric inhibition of FBPase by AMP.