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. 1996 Nov;5(11):2333–2342. doi: 10.1002/pro.5560051120

Evidence for an active T-state pig kidney fructose 1,6-bisphosphatase: interface residue Lys-42 is important for allosteric inhibition and AMP cooperativity.

G Lu 1, B Stec 1, E L Giroux 1, E R Kantrowitz 1
PMCID: PMC2143295  PMID: 8931152

Abstract

During the R-->T transition in the tetrameric pig kidney fructose-1,6-bisphosphatase (Fru-1,6-P2ase, EC 3.1.3.11) a major change in the quaternary structure of the enzyme occurs that is induced by the binding of the allosteric inhibitor AMP (Ke HM, Liang JY, Zhang Y, Lipscomb WN, 1991, Biochemistry 30:4412-4420). The change in quaternary structure involving the rotation of the upper dimer by 17 degrees relative to the lower dimer is coupled to a series of structural changes on the secondary and tertiary levels. The structural data indicate that Lys-42 is involved in a complex set of intersubunit interactions across the dimer-dimer interface with residues of the 190's loop, a loop located at the pivot of the allosteric rotation. In order to test the function of Lys-42, we have replaced it with alanine using site-specific mutagenesis. The kcat and K(m) values for Lys-42-->Ala Fru-1,6-P2ase were 11 s-1 and 3.3 microM, respectively, resulting in a mutant enzyme that was slightly less efficient catalytically than the normal pig kidney enzyme. Although the Lys-42-->Ala Fru-1,6-P2ase was similar kinetically in terms of K(m) and kcat, the response to inhibition by AMP was significantly different than that of the normal pig kidney enzyme. Not only was AMP inhibition no longer cooperative, but also it occurred in two stages, corresponding to high- and low-affinity binding sites. Saturation of the high-affinity sites only reduced the activity by 30%, compared to 100% for the wild-type enzyme. In order to determine in what structural state the enzyme was after saturation of the high-affinity sites, the Lys-42-->Ala enzyme was crystallized in the presence of Mn2+, fructose-6-phosphate (Fru-6-P), and 100 microM AMP and the data collected to 2.3 A resolution. The X-ray structure showed the T state with AMP binding with full occupancy to the four regulatory sites and the inhibitor Fru-6-P bound at the active sites. The results reported here suggest that, in the normal pig kidney enzyme, the interactions between Lys-42 and residues of the 190's loop, are important for propagation of AMP cooperativity to the adjacent subunit across the dimer-dimer interface as opposed to the monomer-monomer interface, and suggest that AMP cooperativity is necessary for full allosteric inhibition by AMP.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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