Análisis Interno de la granja avícola Matilde Esther

The pentose phosphate pathway (PPP) is a key metabolic pathway with two main roles in the cell: it produces reduced NADPH and five carbon sugars, particularly ribose-based. These two roles are spread across two branches of reactions, the oxidative reactions and the non-oxidative reactions [Cabezas et al., 1999]. The PPP is also sometimes known as the phosphogluconate pathway or the hexose monophosphate shunt.

The non-oxidative reactions are primarily used to re-arrange carbon frameworks and convert five-carbon sugars to six-carbon sugars (or vice-versa) via a series of intermediates. All reactions in the non-oxidative phase are completely reversible, indeed, they are driven in “reverse” during the Calvin cycle as part of photosynthesis [Kruger and von Schaewen, 2003]. Importantly for this thesis, two reactions in the non-oxidative pathway are catalysed by the TPP-dependent enzyme transketolase (Figure 2.7). The oxidative reactions are used to generate reduced NADPH. They start with hexose sugar phosphates and convert them to ribose-5-phosphate yielding two moles of reduced NADPH and one mole of CO2for each mole of sugar passed down the pathway. These pentoses can then be re-converted back to hexoses by the non-oxidative branch if required. The overall reaction for the oxidative pathway is:

Glc-6-phosphate + 2 NADP+ + H2O→Rib-5-phosphate + 2 NADPH + 2 H+

The overall reaction for the non-oxidative branch is:

6 Rib-5-phosphate*₎5 Glc-6-phosphate

The first role of the PPP, as a producer of reduced NADPH in the cytosol, is critical in maintaining cellular antioxidant capabilities since reduced NADPH is a substrate for glutathione reductase, the enzyme which maintains the cellular glutathione pool in the reduced state. It also has secondary implications since NADPH is also used in metabolic processes including fatty acid synthesis and sterol synthesis: two molecules of NADPH are oxidised for the addition of each acetyl unit to a growing fatty acid chain. This role of the pathway producing NADPH is critical to cell survival, particularly in erythrocytes which have an active PPP owing to their lack of oxidative metabolism. Additionally, the ribose sugars produced by the pathway can be used as precursors for nucleotide and nucleic acid biosynthesis.

Figure 2.7: Schematic summary of the the pentose phosphate pathway. A:The oxidative and non-oxidative reaction pathways. All reactions in the non-oxidative part are freely reversible, as in the Calvin cycle. Single arrows are only used to indicate flux through the pathway during NADPH synthesis. The number of carbons in each sugar is indicated by the number of sides to the polygon. R5P: Ribose-5-phosphate; S7P: Sedoheptulose-7-phosphate; F6P: Fructose-6-phosphate; G6P: Glucose-6-phosphate; X5P: Xylulose-5-phosphate; G3P: Glyceraldehyde-3-phosphate; E4P: Erythrose-4-phosphate. B: The complete conversion of 6 riboses to 5 hexoses is shown. This is two sets of non-oxidative reactions from A in a mirror-image arrangement.

2.6.1

Control of the pentose phosphate pathway

The first oxidative reaction in the PPP, the conversion of glucose-6-phosphate (G-6-P) to 6- phosphoglucono-δ-lactone, is essentially irreversible and provides the most control over flux through this pathway. It is catalysed by the enzyme glucose-6-phosphate dehydrogenase. Genetic defects in this enzyme can have serious consequences such as the disease favism which is an intolerance to fava beans. Fava beans contain a molecule called divicine which raises cellular H2O2 to fatal levels when G6P dehydrogenase is inactive.

The limiting factor for flux through the pathway is the availability of the oxidised NADP+_{. The NADP}+_{:NADPH ratio in the liver of a well fed rat is around 0.014 mean-} ing there is a vast excess of the reduced NADPH. This is considerably lower than the NAD+_{:NADH ratio in the same tissue which is around 700. Thus, NADPH is only pro-} duced as fast as it is required. In the liver or adipose tissue this rate is higher than it would be in muscle due to the high rate of lipogenesis and thus the high demand for NADPH. The non-oxidative branch of the pathway is primarily controlled by substrate availability and will flow readily in either direction.

This arrangement of oxidative and non-oxidative pathways allows for some interest- ing biochemistry if the cellular need arises. It is possible for cells to use the pathway in one of four ways dependent on the need for ribose-5-phosphate, NADPH and ATP. Firstly, if a cell requires much more R-5-P than NADPH—perhaps a dividing cell synthesising nucleotides—then G-6-P can be converted to fructose-6-phosphate and glyceraldehyde-3- phosphate by normal glycolytic means. Then transketolase and transaldolase can convert these to ribose-5-phosphate with the net result:

5 G-6-P + ATP −→6 R-5-P + ADP + H+

The second mode would be if the needs for NADPH and ribose-5-phosphate are balanced. In this case, the oxidative pathway can be used on its own to produce two moles of NADPH and one mole of ribose-5-phosphate for each mole of glucose-6-phosphate:

G-6-P + 2 NADP+ _{+ H}

2O−→R-5-P + 2 NADPH + 2 H+ + CO2

The third and fourth modes are similar in that the cell requires much more NADPH than ribose-5-phosphate but they differ in whether the cell requires ATP or not. If the cell does not require ATP, the glucose-6-phosphate can be completely oxidised by the PPP yielding only NADPH. If the cell requires ATP, the ribose-5-phosphate can be returned to glycolysis as pyruvate via the non-oxidative branch. This pyruvate can then be used to

either generate ATP or as a precursor for biosynthetic pathways. Both the oxidative and non-oxidative reactions are required here and the metabolites will move in a circular motion (anticlockwise on Figure 2.7 A). The reaction for option three is:

G-6-P + 12 NADP+ _{+ 7 H2O−→6 CO2 + 12 NADPH + 12 H}+ _{+ P}

i

and for option four:

3 G-6-P + 6 NADP+ _{+ 5 NAD}+ _{+ 5 P}

i + 8 ADP−→

5 pyruvate + 3 CO2 + 6 NADPH + 5 NADH + 8 ATP + 2 H2O + 8 H+

These varied options make the PPP incredibly flexible and able to respond to cellular needs in a way that other pathways are unable to. The fact that it provides an alternative route for complete oxidation of glucose may be useful in the diabetic state, especially since diabetes is a state associated with an increase in oxidative stress and the extra NADPH generated by option three above may be useful. “Normal” oxidation of this glucose through the citric acid cycle would primarily generate ATP or increase the mitochondrial membrane potential, exacerbating the production of superoxide.

2.6.2

Transketolase

Transketolase (E.C. 2.2.1.1, TK) is a ubiquitous thiamine pyrophosphate-dependent en- zyme found in all domains of life. It is a homodimer with each chain having a mass of around 68 kDa in humans. There are two active sites within each TK homodimer, each at the interface between the sub-units. Each active site requires one calcium ion and one molecule of TPP as cofactors. It catalyses two reactions within the pentose phos- phate pathway. These are the interconversion of the two pentose phosphates ribose-5- phosphate and xylulose-5-phosphate with the seven-sugar sedoheptulose-7-phosphate and the triosephosphate glyceraldehyde-3-phosphate along with the interconversion of the four- carbon erythrose-4-phosphate and xylulose-5-phosphate with fructose-6-phosphate and gly- ceraldehyde-3-phosphate:

R-5-P + X-5-P*₎S-7-P + GA3P

E-4-P + X-5-P*₎F-6-P + GA3P

The dependence of TK on TPP and a metal ion was originally demonstrated in yeast. TPP can be removed from mammalian TK with relatively mild acidic conditions whilst the

removal of TPP from yeast andE. coli TK requires alkaline conditions [Datta and Racker, 1961] after which the activity is completely ablated [Saitou et al., 1974]. Only after the addition of TPP and a divalent metal ion is activity restored [Heinrich et al., 1972]. This dependence on TPP has also been demonstrated by the presence of TPP in solved crystal structures in yeast [Lindqvist et al., 1992; Nikkola et al., 1994]. The binding of TPP to TK is a two-step process [Esakova et al., 2005; Kochetov and Izotova, 1973]. The first is the binding of the TPP to the apo-enzyme in an fast and readily reversible step yielding a catalytically inactive intermediate. This is followed by a second, slow step which includes conformational changes in the apo-enzyme yielding the active holo-enzyme [Dalby et al., 2007]. In addition, TK is prone to removal of the TPP moiety when any excess is removed [Mitra et al., 1998]. This poses potential problems for the activity of TK in situations where thiamine is deficient.

The mechanism of catalysis in TK is similar to that of PDH in that there is an activated aldehyde being transferred to an acceptor which in the case of TK is an aldose and in the case of PDH is the lipoamide. In both reactions it is the C2 atom of TPP which ionises to yield the carbanion which attacks the carbonyl group of the substrate and forms an activated glycoaldehyde. This then condenses with the new aldehyde to form a ketose sugar which is released from the enzyme [Schenk et al., 1998].