A Switch in the Kinase Domain of Rat Testis 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase^,

Abstract

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase plays an essential role in the regulation of glucose metabolism by both producing and degrading Fru-2,6-P(2) via its distinct catalytic activities. The 6-PF-2-K and Fru-2,6-P(2)ase active sites are located in separate domains of the enzyme. The kinase domain is structurally related to the superfamily of mononucleotide binding proteins that includes adenylate kinase and the G-proteins. We have determined three new structures of the enzymatic monomer, each with a different ligand in the ATP binding site of the 6-PF-2-K domain (AMP-PNP, PO(4), and water). A comparison of these three new structures with the ATPgammaS-bound 6-PF-2-K domain reveals a rearrangement of a helix that is dependent on the ligand bound in the ATP binding site of the enzyme. This helix motion dramatically alters the position of a catalytic residue (Lys172). This catalytic cation is analogous to the Arg residue donated by the rasGAP protein, and the Arg residue at the core of the GTP or GDP sensing switch motion seen in the heterotrimeric G-proteins. In addition, a succinate molecule is observed in the Fru-6-P binding site. Kinetic analysis of succinate inhibition of the 6-PF-2-K reaction is consistent with the structural findings, and suggests a mechanism for feedback inhibition of glycolysis by citric acid cycle intermediates. Alterations in the 6-PF-2-K kinetics of several proteins mutated near both the switch and the succinate binding site suggest a mode of communication between the ATP- and F6P binding sites. Together with these kinetic data, these new structures provide insights into the mechanism of the 6-PF-2-K activity of this important bifunctional enzyme.

Crystal Structure of the Hypoxia-inducible Form of 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3)

“…This conformational change is transmitted to the Fru-6-P loop via Arg-181 such that the Fru-6-P loop has a new conformation. Fru-6-P can bind only to this new conformation (41). Thus, binding of Fru-6-P is dependent on that of ATP in the testis form in an ordered fashion.…”

“…has binding sites for the two substrates, ATP and Fru-6-P, the binding of which are sensitive to the conformations of the two outer lobes known as the ATP loop (residues 168 -179) and the Fru-6-P loop (residues 72-84) that surround the whole active pocket (31,41). In the testis form, the two loops are structurally coupled to each other through two hydrogen bonds donated by Arg-181.…”

Tissue-specific Structure/Function Differentiation of the Liver Isoform of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase

“…A possibility of an effect of phosphorylation of Ser-32 at the mobile N terminus is ignorable since the enzyme was expressed in bacterium and purified in the absence of any protein phosphatase inhibitor such as fluoride. To test its relevance to liganding states and crystal contacts, the three structures of the testis isoform all in different crystal contacts and liganding states (ATP␥S, AMP-PNP, and P i ) (7,9,28) and the three structures of the liver form in three different liganding states (ATP␥S, ADP, and P i ) were cross-compared by superimposing onto each other. The positional differences of C␣s were calculated and presented as Table III.…”

“…In the testis isozyme, it was shown that the last turn of helix ␣5 (residues 162-175) undergoes unwinding upon binding of ATP to recruit Lys-172 to the bridge oxygen between the ␤-and ␥-phosphates of the bound ATP (9,29). This ATP-driven conformational change has been suggested to be transmitted to the Fru-6-P loop via the loop coupling composed of the salt bridges between Asp-177, Arg-78, and Arg-193 and the hydrogen bonds between Arg-181 and the carbonyl oxygens of Tyr-85 and Lys-86 (9,29). Thus, the ATP binding was suggested to be a prerequisite for the subsequent binding of Fru-6-P to the kinase domain in the testis kinase reaction, following the ordered binding of substrates.…”

“…The determination of crystal structures of the rat liver Fru-2,6-P 2 ase domain (6) and the rat testis 6-Fru(P)-2-kinase/Fru-2,6-P 2 ase (7) has led a great progress in our understanding of the catalytic mechanisms of this enzyme system (8,9). However, one of the most fundamental questions about this enzyme system has remained unanswered.…”