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  • However at variance with the

    2021-11-22

    However, at variance with the well-studied R form of FBP1 which is flat, the R state of FBP2 is diametrically different, with a perpendicular orientation of the upper and lower dimers (Barciszewski et al., 2016). The cruciform-like R state of FBP2 is stabilized by a unique hydrophobic motif called ‘leucine lock’, which is formed by leucine residues located within the N-terminal region of FBP2 (L11 and L13) and L190 of one monomer (e.g. C1) and the same residues from another subunit (e.g. C3). In Amrubicin mg to FBP1, both the L13 residue and the N-terminal region are highly conserved in all FBP2 and it has been shown that mutations in this region affect the susceptibility of FBP2 to AMP inhibition (Gizak et Amrubicin mg al., 2008) having only minor effect on FBP1 (Nelson et al., 2001). This in line with observation that AMP association to FBP2 induces structural rearrangement of the N-terminal region from partially disordered (and beta strand-like) to helical structure (Barciszewski et al., 2016). Evidently, the active R-state of FBP2 is less stable than the T-state and also the R- and T-states of FBP1, which is reflected by lower thermal stability of the state (Barciszewski et al., 2016). As in the case of Ca2+-sensitivity the ectotherm FBP2 appears to be less (3–10 times) prone to AMP inhibition than the isozyme from endothermal animals. Kinetic properties of FBP1 from all groups of animals are similar. Additionally, phylogenetic analyses have revealed that vertebrate FBP2 evolved significantly faster than FBP1 (Gizak et al., 2012a, b). To add more complications to the issue of cellular role of the FBPase isozymes, a recent study performed using analytical centrifugation method has demonstrated that in conditions mimicking physiological, FBP2 exists in solution as a mixture of tetramers, dimers and monomers and that addition of AMP induces tetramerisation and inhibition of the isozyme (Wiśniewski et al., 2017). A mutation of L190, the crucial part of the ‘leucine lock’, results in production of dimeric forms only. Such form of FBP2 is fully active but practically insensitive to AMP inhibition (Wiśniewski et al., 2017). This dimeric FBP2 has been overlooked for such a long time probably because in high concentrations of the proteins used for crystallographic studies, both FBP2 and FBP1 are strictly tetrameric.
    Unveiling cellular functions of FBPase in muscle tissue For years, it has been a common belief that lactate produced in glycolysis in a contracting muscle is transported via the blood stream to the liver where it is converted to glucose, which is subsequently transported back to the muscle (’‘the Cori cycle’‘). However, evidence has accumulated that in skeletal muscle up to 50% of lactate is converted to glycogen (Fournier et al., 2002). This suggests that FBP2, the enzyme catalysing a crucial reaction of glyco- and gluconeogenesis simply must be active. However, kinetic studies have revealed that free FBP2 is completely inhibited by physiological concentration of AMP (Skalecki et al., 1995; Rakus and Dzugaj, 2000). In the late 70s, it has been demonstrated that FBPases can interact with aldolases, glycolytic enzymes catalysing both the cleveage of FBP and its synthesis from triose phosphates (Pontremoli et al., 1979; MacGregor et al., 1980), but the meaning of such interaction has remained unknown up to this millennium. The studies using muscle proteins have revealed that FBP2 is entirely desensitised to AMP by interactions with muscle isoform of aldolase, ALDOA (Rakus and Dzugaj, 2000; Rakus et al., 2003b). The latter studies have demonstrated that FBP2 and ALDOA form, in a metabolite-dependent manner, a metabolic complex (Rakus et al., 2003b) in which a substrate channelling phenomenon occurs (Rakus et al., 2004). Substrate channelling is a process in which the intermediate produced by one enzyme is directly transferred to the next enzyme, without complete equilibration with the bulk phase. It is assumed that substrate channelling between ALDOA and FBP2, precluding diffusion of the intermediate into the bulk phase and thereby protecting FBP against its degradation by glycolytic pathway (precisely: by another pool of ALDOA), is the elementary mechanism enabling glycogen synthesis from carbohydrate precursors in muscle fibres (Rakus et al., 2004).