Binding of AMP to two of the four subunits of pig kidney fructose-1,6-bisphosphatase induces the allosteric transition

To study the allosteric transition in pig kidney fructose 1,6-bisphosphatas e (FBPase), we constructed hybrids in which subunits have either their acti ve or regulatory sites rendered nonfunctional by specific mutations. This w as accomplished by the coexpression of the enzyme from a plasmid that conta ined two slightly different copies of the cDNA. To resolve and purify each of the hybrid enzymes, six aspartic acid codons were added before the termi nation codon of one of the cDNAs. The addition of these Asp residues to the protein did not alter the kinetic or allosteric properties of the resultin g FBPase. Expression of the enzyme from a dual-gene plasmid resulted in the production of a set of five different enzymes (two homotetramers and three hybrid tetramers) that could be purified by a combination of affinity and anion-exchange chromatography because of the differential charge on each of these species. The hybrid with one subunit that only had a functional regu latory site (R) and three subunits that only had a functional active site ( A) exhibited biphasic AMP inhibition. Analysis of these data suggest that t he binding of AMP to the R subunit is able to globally alter the activity o f the other three A subunits. The hybrid composed of two R and two A subuni ts is completely inhibited at an AMP concentration of approximate to0.5 mM, 100-fold less than the concentration required to fully inhibit the A(4) en zyme. The monophasic nature of this cooperative inhibition suggests that th e AMP binding to the two R subunits is sufficient to completely inhibit the enzyme and suggests that the binding of AMP to only two of the four subuni ts of the enzyme induces the global allosteric transition from the R to the T state. (C) 2001 Wiley-Liss, Inc.