Fructose-2,6-bisphosphate muscle

Fructose bisphosphate aldolase of animal muscle is a Class I aldolase, which forms a Schiff base or imme intermediate between the substrate (fructose-1,6-bisP or dihydroxyacetone-P) and a lysine amino group at the enzyme active site. The chemical evidence for this intermediate comes from studies with the aldolase and the reducing agent sodium borohydride, NaBH4. Incubation of fructose bisphosphate aldolase with dihydroxyacetone-P and NaBH4 inactivates the enzyme. Interestingly, no inactivation is observed if NaBH4 is added to the enzyme in the absence of substrate. 

Callens, M., Kuntz, D. A. and Opperdoes, F. R. (1991) Kinetic properties of fructose bisphosphate aldolase from Trypanosoma brucei compared to aldolases from rabbit muscle and Staphylococcus aureus. Mol. Biochem. Parasitol. 47 1 10. 

Fig. 2 Lactate dehydrogenase a) a ribbon representation of the tetramer of the B. stearothermophilus enzyme with each peptide chain depicted in a different color. The cofactor and oxamate inhibitor are colored according to atom type, as is fructose bisphosphate. which is an allosteric regulator of the enzyme, b) On the left is a detailed view of the enzyme active site as seen in the crystal structure. The ligand is highlighted in green and key amino acid residues are labeled. This is compared with the traditional two-dimensional representation of the enzyme mechanism on the right. Note that the residue numbers differ slightly from those of the muscle enzyme discussed in the test. (View this an i i color at www.dekker.com.)...

Fructose bisphosphate aldolase (isoenzyme B, M, 156,000) (EC 4.1.2.13). Fructosemia, fructosuria and hypoglucosemia after intake of fructose. Intracellular accumulation of fructose 1-phosphate, Hyperurate-mia. Hepatomegaly. Renal tubular dysfunction. Intraocular bleeding. Patients symptom-free and healthy if fructose avoided. Aldolase A (muscle and most other tissues) and aldolase C (brain and heart) present and fully active. 

If alanine accumulates in muscle, it acts as an allosteric inhibitor of pyruvate kinase, so reducing the rate at which pyruvate is formed. This end-product inhibition of pyruvate kinase by alanine is over-ridden by high concentrations of fructose bisphosphate, which acts as a feed-forward activator of pyruvate kinase. ATP is an inhibitor of pyruvate kinase, and at high concentrations acts to inhibit the enzyme. More importantly, ATP acts as an allosteric inhibitor of phosphofructokinase (section 10.2.2.1). This means that, under conditions in which the supply of ATP (which can be regarded as the end-product of all energy-yielding metabolic pathways) is more than adequate to meet requirements, the metabolism of glucose is inhibited. 

The structure of human muscle fructose-1,6-bisphosphate aldolase, as determined by X-ray crystallography and downloaded from the Protein Data Bank. (PDB ID 1ALD Gamblin, S. J., Davies, G. J., Grimes, J. M., Jackson, R. M., Littlechild, J. A., Watson, H. C. Activity and specificity of human aldolases. J. Mol. Biol. v219, pp. 573-576, 1991.)... 

The convetsion of fructose 1,6-bisphosphate to fructose 6-phosphate, to achieve a reversal of glycolysis, is catalyzed by fructose-l,6-bi pho pbatase. Its ptesence determines whether or not a tissue is capable of synthesizing glycogen not only from pymvate but also from ttiosephosphates. It is present in hvet, kidney, and skeletal muscle but is probably absent from heart and smooth muscle. 

In liver, cAMP activates gluconeogenesis, but in muscle, it activates glycolysis. Let s do liver first, and the muscle answer will just be the opposite. So, we want to activate gluconeogenesis in liver in response to increased phosphorylation (increased levels of cAMP). Phosphorylation of our enzyme (PFK-2) must have an effect that is consistent with the activation of gluconeogenesis. If gluconeogenesis is on and glycolysis is off, the level of fructose 2,6-bisphosphate (an activator of glycolysis) must fall. If fructose 2,6-bisphosphate is to fall, the PFK-2 that synthesizes it must be made inactive. So, in liver, phosphorylation of PFK-2 must inactivate the enzyme. 

In muscle, phosphorylation of PFK-2 in response to increased cAMP activates the enzyme, the level of fructose 2,6-bisphosphate rises, and glycolysis is activated. 

There s also a fructose 2,6-bisphosphatase that hydrolyzes fructose 2,6-bisphosphate see if you can figure out what happens to the phosphatase activity in liver and muscle when the enzyme is phosphorylated. As a check to your answer, you might recall the PFK-2 and fructose 2,6-bisphosphatase are one and the same protein. Phosphorylation-dephosphorylation actually shifts the activity of this single protein between the kinase and the phosphatase. So the answer you get should be opposite to the one we got earlier. 

Figure 11-2 Roles of phosphofructose kinase and fructose 1,6-bisphosphatase in the control of the breakdown and storage (—+) of glycogen in muscle. The uptake of glucose from blood and its release from tissues is also illustrated. The allosteric effector fructose 2,6-bisphosphate (Fru-2,6-P2) regulates both phosphofructokinase and fructose 2,6-bisphosphatase. These enzymes are also regulated by AMP if it accumulates. The activity of phosphofructokinase-2 (which synthesizes Fru-2,6-P2) is controlled by a cyclic AMP-dependent kinase and by dephosphorylation by a phosphatase.

The Hormone Epinephrine Stimulates Glucose Production in Both Liver Cells and Muscle Cells The Hormonal Regulation of the Flux between Fructose-6-phosphate and Fructose-1,6-bisphosphate Is Mediated by Fructose-2,6-bisphosphate... 

Fig. 2. Schematic representation of substrate binding and C-C bond formation for the class I fructose 1,6-bisphosphate aldolase from rabbit muscle...

Fig. 8. Stability of the rhamnulose 1-phosphate aldolase from Escherichia coli (RhuA) vs. that of the fructose 1,6-bisphosphate aldolase from rabbit muscle (FruA) in phosphate buffer (pH 7.2 25°C ca. 1 Uml ) a) RhuA b) O RhuA, 30% EtOH c) RhuA, 50% DMSO d) FruA...

In brain tissues, specific isoforms of glycolytic enzymes are also expressed there are specific brain isoforms for PFK (PFK-C), fructose-1,6-bisphosphate aldolase (aldolase C), enolase (enolase y), but not for GAPDH. The isoforms bear the same catalytic functions however, they could be specialized to form different ultrastructural entities. For example, muscle PFK (a dissociable tetrameric form) binds to microtubules and bundle them [94, 95], however, the brain isoenzyme (stable tetramer) does not [96]. 

Furthermore, the catalytic efficiency (K t/KM) of 84G3 for this substrate, 3.3 X 105 s-1M-1, is comparable with the efficiency of natural muscle aldolase, 4.9 X 104 s 1M 1, in the retro-aldolization of its substrate fructose 1, 6-bisphosphate (Morris and Tolan, 1994). However, these two enzymes use different substrates, and the rates were recorded at different temperatures (22°C for 84G3, 4°C for the natural aldolase). Despite this, we believe that it will be possible to develop a catalytic antibody that, under identical conditions, has a faster cat and lower KM than a natural enzyme for the same substrate. 

The use of aldolases and transketolase has opened the way to many highly multifunctional organic compounds [1]. In organic synthesis, the most widely used dihydroxyacetonephosphate (DHAP) aldolase is the commercially available fruc-tose-1,6-bisphosphate aldolase from rabbit muscle (FruA). This enzyme is a key enzyme of the glycolytic pathway, reversibly catalyzing the cleavage of fructose-... 

Uycda, K., Fimiya, E., and Luby, L- J. (1981). The effect of natural and synthetic t>fructose-2/i-bisphosphate on the regulatory kinetic properties of liver and muscle phosphofnic-tokinase. /. Biof. Orem. 2S6,8394 399. 

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