Importance of the Dimer-Dimer Interface for Allosteric Signal Transduction and AMP Cooperativity of Pig Kidney Fructose-1,6-Bisphosphatase: SITE-SPECIFIC MUTAGENESIS STUDIES OF GLU-192 AND ASP-187 RESIDUES ON THE 190'S LOOP

Abstract

The role of the 190's loop of fructose-1,6-bisphosphatase (Fru-1, 6-P2ase) in the allosteric regulation of Fru-1,6-P2ase has been investigated through kinetic studies on three mutant enzymes, Glu-192 --> Ala, Glu-192 --> Gln, and Asp-187 --> Ala. AMP is an allosteric inhibitor, which binds to the regulatory sites and induces the R- to T-state transition; for wild-type Fru-1,6-P2ase AMP inhibition is cooperative with a Hill coefficient of 2.0. The replacement of Asp-187, which forms an interaction across the C1:C2 monomer-monomer interface, with alanine did not change the catalytic efficiency, and it had no effect on the cooperativity of AMP inhibition; however, the apparent dissociation constant for AMP increased more than 4-fold as compared to the value for the wild-type enzyme. The replacement of Glu-192, which forms interactions across the C1:C4 dimer-dimer interface, with Ala and Gln lowered kcat from 21 s-1 for wild-type enzyme to 15 s-1 and 13 s-1, respectively, for the mutant enzymes, while their respective Km values were not changed. However, these replacements did have dramatic effects on AMP inhibition; first, cooperative AMP inhibition was lost; second, the AMP inhibition was biphasic, which can be interpreted as due to AMP binding to two classes of binding sites. The high affinity class of sites corresponds to the regulatory sites, while the low affinity class of sites may be the active sites. The results reported here, combined with the structural and kinetic results from the Lys-42 --> Ala enzyme, strongly suggest that the C1:C4 dimer-dimer interface, rather than the C1:C2 monomer-monomer interface, is critical for the propagation of the allosteric signal between the AMP sites on different subunits; in addition, cooperative AMP inhibition is essential for the enzyme to be fully inhibited by the binding of AMP to the allosteric site.