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

Abstract

The crystal structures of the human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in three different liganding states were determined and compared with those of the rat testis isozyme. A set of amino acid sequence heterogeneity from the two distinct genes encoding the two different tissue isozymes leads to both global and local conformational differences that may cause the differences in catalytic properties of the two isozymes. The sequence differences in a ␤؊hairpin loop in the kinase domain causes a translational shift of several hydrophobic interactions in the dimeric contact region, and its propagation to the domains interface results in a 5°twist of the entire bisphosphatase domain relative to the kinase domain. The bisphosphatase domain twist allows the dimeric interactions between the bisphosphatase domains, which are negligible in the testis enzyme, and as a result, the conformational stability of the domain is increased. Sequence polymorphisms also confer small but significant structural dissimilarities in the substrate-binding loops, allowing the differentiated catalytic properties between the two different tissue-type isozymes. Whereas the polymorphic sequence at the bisphosphatase-active pocket suggests a more suitable substrate binding, a similar extent of sequence differences at the kinase-active pocket confers a different mechanism of substrates bindings to the kinase-active pocket. It includes the ATP-sensitive unwinding of the switch helix ␣5, which is a characteristic ATP-dependent conformational change in the testis form. The sequence-dependent structural difference disallows the liver kinase to follow the ATP-switch mechanism. Altogether these suggest that the liver isoform has structural features more appropriate for an elevated bisphosphatase activity, compared with that of the testis form. The structural predisposition for bisphosphatase activity in the liver isozyme is consistent with the liver-unique glucose metabolic pathway, gluconeogenesis.

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

“…For example, S162e means the 162nd residue in the inducible protein is Ser and the equivalent residue in the liver form is Glu. As predicted from the high sequence homology (85%) between the PFKFB isoforms, the folding of the inducible form is very similar to those of the testis and liver forms (31,37). As shown in Fig.…”

“…The conformations of the substrate loops in the 2-Kase domain are different from those of other isoforms (31,37), providing a structural rationale for the higher 2-Kase activity. The structure of the N-terminal regulatory domain is revealed: the N terminus binds to the 2-Pase domain to cause a local conformational change in the active pocket to enhance inhibitory binding of product.…”

“…Overall Structure-The PFKFB3 protein was crystallized under conditions similar to those for the liver form (31). The structures from the two different liganding conditions were determined by molecular replacement using the liver structure as a start model.…”

“…However, the native liver form with a cysteine instead of an arginine has the two loops decoupled from each other. As a result, their conformations are different from those in the testis form, and the two substrates bind independently from each other (31).…”

“…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.…”