Fructose 2,6-bisphosphate enzymes

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. 

This is interconverted to form glyceraldehyde 3-phosphate and both combine, via the enzyme aldolase, to produce fructose bisphosphate, en route to form glucose or glycogen. 

Fructose-bisphosphate aldolase, which also acts on fructose-1-phosphate, is the enzyme deficient in hereditary fructose intolerance (HFI, MIM 229 600). 

Type I aldolases, which include the most studied mammalian enzymes, have a more complex mechanism involving intermediate Schiff base forms (Eq. 13-36, steps a, V, c, d ).m The best known members of this group are the fructose bisphosphate aldolases (often referred to simply as aldolases), which cleave fructose-1,6-P2 during glycolysis (Fig. 10-2, step e). 

An analogous use of ATP is found in photosynthetic reduction of carbon dioxide in which ATP phos-phorylates ribulose 5-P to ribulose bisphosphate and the phosphate groups are removed later by phosphatase action on fructose bisphosphate and sedoheptulose bisphosphate (Section J,2). Phosphatases involved in synthetic pathways usually have a high substrate specificity and are to be distinguished from nonspecific phosphatases which are essentially digestive enzymes (Chapter 12). 

The same protein kinase that phosphorylates glycogen phosphorylase and glycogen synthase does not phosphorylate the enzymes of pseudocycle II. Rather an enzyme gets phos-phorylated that catalyzes the synthesis of a potent allosteric effector of the two relevant enzymes, phosphofructokinase and fructose bisphosphate phosphatase. In the liver the un-phosphorylated form this enzyme synthesizes fructose-2,6-bisphosphate. Phosphorylation converts it into a degradative enzyme for the same compound. Fructose-2,6-bisphosphate is an activator of phosphofructokinase and an inhibitor of fructose bisphosphate phosphatase. As a result the net effect of glucagon on pseudocycle II is to stimulate fructose bisphosphate phosphatase while inhibiting phosphofructokinase (see table 12.2 and fig. 12.30). 

In proteins with a symmetric structure, circular permutation can account for the shift of active-site residues over the course of evolution. A very good model of symmetric proteins are the (/Ja)8-barrel enzymes with their typical eightfold symmetry. Circular permutation is characterized by fusion of the N and C termini in a protein ancestor followed by cleavage of the backbone at an equivalent locus around the circular structure. Both fructose-bisphosphate aldolase class I and transaldolase belong to the aldolase superfamily of (a/J)8-symmetric barrel proteins both feature a catalytic lysine residue required to form the Schiff base intermediate with the substrate in the first step of the reaction (Chapter 9, Section 9.6.2). In most family members, the catalytic lysine residue is located on strand 6 of the barrel, but in transaldolase it is not only located on strand 4 but optimal sequence and structure alignment with aldolase class I necessitates rotation of the structure and thus circular permutation of the jS-barrel strands (Jia, 1996). 

D-fructose-6-phosphate + ATP <=> D-fructose-l,6-bisphosphate Enzyme Phosphofructokinase... 

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-1,6-Pj v dihydroxy-acetone-P + glyceraldehyde-3-P Fructose-bisphosphate aldolase (EC 4.1.2.13) Chelating agents (only + 24.0 with bacterial enzymes) (-I- 5.73) ... 

The erythrose 4-phosphate generated in the same step (Scheme 11.7) as the derivative of thiamine diphosphate is then available for enzyme-catalyzed aldol-type condensation with dihydroxyacetone monophosphate just as shown in Scheme 11.5 for the analogous reaction with glyceraldehyde 3-phosphate and using the same fructose-bisphosphate aldolase (EC 4.1.2.13). 

Scheme 11.10. A representation of the aldol-type condensation of erythrose 4-phosphate with dihydroxyacetone monophosphate using fructose bisphosphate aldolase (EC 4.1.2.13) to produce a seven-carbon sugar, sedoheptulose 1,7-bisphosphate. The enzyme-catalyzed hydrolysis (EC 3.1.3.37) yielding sedoheptulose 7-phosphate is also shown.

Fructose bisphosphate is hydrolysed to fructose 6-phosphate by a simple hydrolysis reaction catalysed by the enzyme fructose bisphosphatase. 

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. 

A substrate cycle is produced by the simultaneous operation of opposing but chemically distinct reactions (32). For example, the enzymes 6-phosphofructokinase (PFK) and fructose bisphosphatase (FBPase) catalyze nonequilibrium and opposing reactions so that they can produce the fructose 6-phosphate/fructose bisphosphate substrate cycle, as follows ... 

Since is positive, cycling will increase the sensitivity of the flux to regulator X provided that s j>r ,), that is, if the sensitivity of the subsequent reaction of the system to the concentration of the product, P, is greater than the sensitivity of the reverse reaction of the cycle to the concentration of P. This situation can be met in several ways (9). For example, in the fructose 6-phosphate/fructose bisphosphate cycle, fructose bisphosphatase (equivalent to C above) has aK for fructose 1,6-bis-phosphate of about 1 pM (or at least two orders of magnitude lower than many other glycolytic enzymes for their glycolytic substrates) so that it is probably saturated with fructose bisphosphate in vivo (equivalent to P in... 

In the splitting stage, fructose 1,6-bisphosphate, a six-carbon molecule, is cleaved into two three-carbon molecules, glycerone phosphate (also called dihydroxyacetone phosphate) and glyceraldehyde 3-phosphate, by the enzyme fructose-bisphosphate aldolase. The name of the enzyme is derived from... 

Figure 6.24 The function of the enzyme phosphofructokinase. (a) Phosphofructokinase is a key enzyme in the gycolytic pathway, the breakdown of glucose to pyruvate. One of the end products in this pathway, phosphoenolpyruvate, is an allosteric feedback inhibitor to this enzyme and ADP is an activator, (b) Phosphofructokinase catalyzes the phosphorylation by ATP of fructose-6-phosphate to give fructose-1,6-bisphosphate. (c) Phosphoglycolate, which has a structure similar to phosphoenolpyruvate, is also an inhibitor of the enzyme.

FIGURE 19.9 Fructose-2,6-bisphosphate activates phosphofructokinase, iucreasiug the affinity of the enzyme for fructose-6-phosphate and restoring the hyperbolic dependence of enzyme activity on substrate. 

Pyruvate kinase possesses allosteric sites for numerous effectors. It is activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine. (Note that alanine is the a-amino acid counterpart of the a-keto acid, pyruvate.) Furthermore, liver pyruvate kinase is regulated by covalent modification. Flormones such as glucagon activate a cAMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the enzyme. The phos-phorylated form of pyruvate kinase is more strongly inhibited by ATP and alanine and has a higher for PEP, so that, in the presence of physiological levels of PEP, the enzyme is inactive. Then PEP is used as a substrate for glucose synthesis in the pathway (to be described in Chapter 23), instead... 

The hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate (Eigure 23.7), like all phosphate ester hydrolyses, is a thermodynamically favorable (exergonic) reaction under standard-state conditions (AG° = —16.7 kj/mol). Under physiological conditions in the liver, the reaction is also exergonic (AG = —8.6 kJ/mol). Fructose-1,6-bisphosphatase is an allosterically regulated enzyme. Citrate stimulates bisphosphatase activity, hut fructose-2,6-bisphosphate is a potent allosteric inhibitor. / MP also inhibits the bisphosphatase the inhibition by / MP is enhanced by fructose-2,6-bisphosphate. 

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