Fructose-1,6-diphosphate, glycolysis

It is now generally recognized that an important site of regulation of both glycolysis and gluconeogenesis is at the level of fructose diphosphate formation and hydrolysis (10). In the direction of glycolysis, the activity of phosphofructokinase is inhibited by ATP and citrate, and this inhibition is reversed by AMP (11). The discovery that FDPase... 

Fig. 1 0.3 A simple model of the reaction mechanism of glycolysis. For definition of symbols see fig. 6.1. FDP, fructose diphosphate PYR, pyruvate LAC, lactate. (From [11].)...

Its role in glycolysis is obvious, since it is responsible for the equilibration between the two triose units derived from the cleavage of fructose diphosphate by aldolase. The enzyme occurs in large amounts in skeletal muscle as well as in malignant tumors, brain, and yeast. Although Meyerhof and Beck have purified it some thirty times, this enzyme has not been extensively studied. The TN is enormous—some 1,000,000 per 10 g. protein. 

This cleavage is a retro aldol reaction It is the reverse of the process by which d fruc tose 1 6 diphosphate would be formed by aldol addition of the enolate of dihydroxy acetone phosphate to d glyceraldehyde 3 phosphate The enzyme aldolase catalyzes both the aldol addition of the two components and m glycolysis the retro aldol cleavage of D fructose 1 6 diphosphate... 

Figure 17-2. The pathway of glycolysis. ( ,—P, HOPOj " .inhibition.) At asterisk Carbon atoms 1-3 of fructose bisphosphateform dihydroxyacetone phosphate, whereas carbons 4-6 form glyceraldehyde 3-phosphate. The term "bis-," as in bisphosphate, indicates that the phosphate groups are separated, whereas diphosphate, as in adenosine diphosphate, indicates that they are joined.

Phosphofructokinase (PFK) is a key regulatory enzyme of glycolysis that catalyzes the conversion of fructose-6-phosphate to fructose-1,6-diphosphate. The active PFK enzyme is a homo- or heterotetrameric enzyme with a molecular weight of 340,000. Three types of subunits, muscle type (M), liver type (L), and fibroblast (F) or platelet (P) type, exist in human tissues. Human muscle and liver PFKs consist of homotetramers (M4 and L4), whereas red blood cell PFK consists of five tetramers (M4, M3L, M2L2, ML3, and L4). Each isoform is unique with respect to affinity for the substrate fructose-6-phosphate and ATP and modulation by effectors such as citrate, ATP, cAMP, and fructose-2,6-diphosphate. M-type PFK has greater affinity for fructose-6-phosphate than the other isozymes. AMP and fructose-2,6-diphosphate facilitate fructose-6-phosphate binding mainly of L-type PFK, whereas P-type PFK has intermediate properties. 

Pyruvate kinase (PK) is one of the three postulated rate-controlling enzymes of glycolysis. The high-energy phosphate of phosphoenolpyruvate is transferred to ADP by this enzyme, which requires for its activity both monovalent and divalent cations. Enolpyruvate formed in this reaction is converted spontaneously to the keto form of pyruvate with the synthesis of one ATP molecule. PK has four isozymes in mammals M, M2, L, and R. The M2 type, which is considered to be the prototype, is the only form detected in early fetal tissues and is expressed in many adult tissues. This form is progressively replaced by the M( type in the skeletal muscle, heart, and brain by the L type in the liver and by the R type in red blood cells during development or differentiation (M26). The M, and M2 isozymes display Michaelis-Menten kinetics with respect to phosphoenolpyruvate. The Mj isozyme is not affected by fructose-1,6-diphosphate (F-1,6-DP) and the M2 is al-losterically activated by this compound. Type L and R exhibit cooperatively in... 

Two metabolic patterns are discernible from the results. Carbon atoms 2, 1, and 7 of shikimate (VI) are derived almost equally from G-1,6, G-2,5, and G-3,4, respectively. In the Embden-Meyerhof pathway of hexose metabolism (see Fig. 2), D-fructose 1,6-diphosphate is cleaved to 1,3-dihydroxy-2-propanone phosphate (G-1,2,3) and D-glycerose 3-phosphate (G-4,5,6), and the two trioses are interconverted by triose phosphate isomerase. The observed randomization of label between Cl and C6, C2 and C5, and C3 and C4 of hexose therefore implies that C2, Cl, and C7 of shikimate are derived from a 3-carbon intermediate of glycolysis. The small but significant preponderance of G-6 over G-1, of G-5 over G-2, and, presumably, of G-4 over G-3, can be explained by recent observations that, in the aldolase cleavage of D-fructose 1,6-diphosphate, the 1,3-dihy-... 

The conversion, by bacterial extracts, of D-oZtro-heptulose 1,7-diphosphate to shikimate, essentially without side reactions, greatly facilitated subsequent study of the intermediate steps in the synthesis. It was shown that the addition of iodoacetate or fluoride completely blocks this conversion. In the presence of iodoacetate, synthesis is restored by the addition of either D-glyceronic acid 3-phosphate or enolpyruvate phosphate. In the presence of fluoride, only enolpyruvate phosphate is able to restore shikimate synthesis. Neither D-fructose 1,6-diphosphate nor pyruvate reverses these inhibitions. These results suggested that the reactions of glycolysis, from triose phosphate to enolpyruvate phosphate (see Fig. 2), are involved in the conversion of D-oZfro-heptulose diphosphate to shikimate. The effect... 

Fructose-6-phosphate formed from the isomerization discussed above is further phos-phorylated during glycolysis to fructose-1,6-diphosphate (108), which is then cleaved by fructose-1,6-bisphosphate aldolase to afford dihydroxy acetone phosphate (109) and glyceraldehyde-3-phosphate (110). This cleavage reaction is the reverse of an aldol condensation discussed in Section II.C and during gluconeogenesis. In the latter case, fructose-1,6-bisphosphate aldolase catalyzes the reverse reaction herein via aldol condensation of the ketose 109 and the aldose 110 to form linear fructose-1,6-bisphosphate (108) . 

Two other allosteric enzyme regulatory reactions also help to regulate glycolysis the conversion of fructose 6-phosphate to fructose 1,6-diphosphate by phos-phofructokinase and the conversion of phosphoenolpyru-vate to pyruvate by pyruvate kinase. 

In the actual cleavage reaction of glycolysis, D-fructose< 1,6-diphosphate is converted into D-glyceraldehyde-3-phosphate and dihydroxyacetone, CH2OHCOCH2OH. What kind of reaction is this, basically Sketch out a possible mechanism, neglecting, of course, the all-important role of the enzyme. (Hints The enzyme required is called aldolase. See Problem 21.14, p. 711.)... 

The reaction is essentially irreversible under physiological conditions and is a major regulatory step of glycolysis. PFK-1 is an inducible, highly regulated, allosteric enzyme. In its active form, muscle PFK-1 is a homotetramer (M.W. 320,000) that requires K+ or NH4, the latter of which lowers Km for both substrates. When adenosine triphosphate (ATP) levels are low during very active muscle contraction, PFK activity is modulated positively despite low concentration of fructose-6-phosphate. Allosteric activators of muscle PFK-1 include adenosine monophosphate (AMP), adenosine diphosphate (ADP), fructose-6-phosphate, and inorganic phosphate (Pi) inactivators are citrate, fatty acids, and ATP. 

The first step of the trypanosomal glycolysis involves the conversion of one molecule of glucose into glucose-6-phosphate, which is transformed to fructose-1,6-diphosphate. The latter is then converted into glyceraldehyde-3-phosphate, PEP and pyruvate (PYR) by a sequence of reactions similar to those described for helminth glycolysis (Chapter 2, Chart 2) [1,3-6],... 

Fructose-1,6-diphosphate aldolase of rabbit muscle has been studied very extensively-and it is now commercially available. That of spinach leaves, obviously very accessible, was recently examined (Valentin and Bolte 1993). In the fundamental reaction of glycolysis (reaction 6.8), the donor is dihydroxyacetone phosphate. It can scarcely be varied, but there is more flexibility with the acceptor (David et al. 1991 Bednarski et al. 1989), and sometimes we can wander considerably from the subject of sugar chemistry. In any case, the vicinaZ-diol created at positions 3 and 4 (uloses numbering) has the D-threo configuration. Hence the condensation of the keto aldehyde 6.28 gives ketose 6.29 which, after isolation, is dephosphorylated enzymically in the presence of acid phosphatase. 

The substrate for the first enzyme-catalyzed reaction of glycolysis is a six-carbon compound (o-glucose). The final product of glycolysis is two molecules of a three-carbon compound (pyruvate). Therefore, at some point in the series of enzyme-catalyzed reactions, a six-carbon compound must be cleaved into two three-carbon compounds. The enzyme aldolase catalyzes this cleavage (Figure 24.12). Aldolase converts o-fructose-l,6-diphosphate into o-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. The enzyme is called aldolase because the reverse reaction is an aldol addition reaction (Section 19.13). The reaction proceeds as follows ... 

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