Pentose Phosphate Pathway History of

Alive after the Fall Review

Surviving World War III

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John F. Williams

Australian National University, Canberra, Australia

In this article the oxidative and non-oxidative segments of the pentose pathway (PP) of glucose metabolism are defined. The discovery in 1931-1935 by the German biochemist, Otto Warburg, of the oxidative division of the pathway and of the chemistry and role of a new pyridine nucleotide co-enzyme in its reactions is deliniated.

Background

The successful revelation of many of the reactants and enzymes of the non-oxidative PP was largely achieved by two American biochemists, Bernard L. Horecker and Ephraim Racker who, during 1950-1955, independently accomplished our current text-book knowledge of the classical reaction scheme of PP. A precise reaction order and metabolic map depiction of PP reactions was published by Horecker in 1955 from results of prediction labeling experiments that used variously labeled 14C-ribose 5-phosphate dissimilation by liver and plant tissue extracts. Many PP reactions were also assigned roles in the path of carbon in photosynthesis that was unraveled by Calvin and colleagues during 1945-1954. The above accomplishments briefly terminated fundamental research on the nature of PP which was then replaced by an era (1958-1979) of metabolic pathway measurement.

Joseph Katz and Harland G. Wood were the pioneers who developed theory and methods for quantitative measurement of the contributions of pentose cycle (PC) and other pathways to total glucose metabolism. All of these methods depended on the metabolism of 14C-labeled substrates. PC was measured to a significant level in many animal tissues but made its most notable contribution in adipose tissue, which resulted in the PP being renamed the (fat) F-type PP. Contrary to all expectation, many attempts to quantify PC in liver, the major tissue used by Horecker to establish the reaction scheme for the F-type PP, showed it measured a negligible and therefore puzzling contribution to glucose metabolism.

Based on these latter findings the reaction mechanism of PP was reinvestigated during 1965-1992 by Williams and colleagues in Australia. Liver featured in the investigations because it is a rich source of the enzymes and individual reactions of PP. New octulose and heptulose phosphate reactants and new enzyme activites were discovered. Using specifically 14C-labeled substrates, a revised (liver) L-type PP was identified which quantitatively accounted for 25% of total glucose metabolism. These results aroused criticism and polemic by those defending F-type PC theory and measurement practice. This unsettling state was put to rest in 1993 when the Williams group, using 13C-NMR spectrometry showed that the three group transferring enzymes of the PP, namely transketolase, transaldolase and aldolase catalyzed simultaneous mass transfer and group exchange reactions and in all cases the exchange rates exceeded the mass flux rate. As an example it was found that Transketolase exchange rates in normal and regenerating liver, four different neoplasms, spinach chloroplasts and adipocytes exceeded PP flux rates by

5-600 times. Since the groups being transferred by the above enzymes are 14C-labeled in all of the unraveling investigations that involved F-type and L-type pathways, it is concluded that carbon isotope prediction labeling data are useless for the investigations of the reaction order and measurement of PP. Thus metabolic maps of PP in textbooks of biochemistry are erroneous. A concluding statement that best describes how the PP should now be viewed is given.

All or at least a substantial selection of the reactions of the pentose phosphate (PP) pathway of glucose metabolism occur in the cytoplasmic compartment of most cells. The reaction scheme is in two parts and consists of oxidative (Figure 1) and nonoxidative segments (Figure 2). The reactions of the oxidative segment are few and involve the decarboxylation of glucose-6-phosphate (Glc 6-P) to ribulose 5-phosphate (Ru 5-P) via 6-phosphogluconolactone and 6-phosphogluconate (6-Pg). There is concomitant production of two moles each of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and proton for each mole of Glc

6-P converted to Ru 5-P by oxidative decarboxylation. The non-oxidative reactions (Figure 2) are classically

[2-c]glucose

FIGURE 1 Reactions of the oxidative segment of the pentose pathway showing the conversion of [2-14C]-glucose to CO2 and [1-14C]-ribulose 5-phosphate. Glucose is phosphorylated by hexokinase and oxidatively decarboxylated by the concerted actions of glucose 6-phosphate dehydrogenase, water at pH 7.4 or lactonase and 6-phosphogluconate dehydrogenase (see glossary for equation).

[2-c]glucose

FIGURE 1 Reactions of the oxidative segment of the pentose pathway showing the conversion of [2-14C]-glucose to CO2 and [1-14C]-ribulose 5-phosphate. Glucose is phosphorylated by hexokinase and oxidatively decarboxylated by the concerted actions of glucose 6-phosphate dehydrogenase, water at pH 7.4 or lactonase and 6-phosphogluconate dehydrogenase (see glossary for equation).

depicted as a diversity of reversible steps for the interconversion and linkage of other pentose phosphate products that arise from Ru 5-P, with triose and hexose phosphates that are also common intermediates in cytoplasm of the higher flux glycolytic pathway of glucose metabolism. The reactions of Figure 2 also show sugar phosphate intermediates (glycolyl units) with 3, 4, 5, 6, and 7 carbon atoms that are generated by the reversible reactions of the nonoxidative segment. There are thus three biosynthetic functions of PP reactions that relate to cellular energetics, growth, and repair: (1) the contribution by the oxidative segment to the provision and maintenance of a high NADPH/ NADP+ redox potential and thereby the supply of electrons for some reductive anabolic processes; (2) the formation of ribose 5-phosphate (Rib 5-P) for all nucleotide and nucleic acid biosynthesis; and (3) a storage pool of diverse phosphorylated glycolyl units that may be used in biosynthetic and energy-yielding reactions by other pathways. Finally, selected reactions of the nonoxidative segment are also part of the most extensive synthetic and life-sustaining event on the planet, namely, the photosynthetic reductive path of CO2 assimilation in all C-3 plants.

Early Discoveries

A summary of the biochemistry of the reactions, enzymes, methods, distributions, and quantitation of the pathways of Figures 1 and 2 in tissues, together with all references cited in this article that relate to the history of the "unraveling" events of the pathway, are given in a comprehensive review of the PP by Williams J. F. et al. in 1987. Discoveries which led to the formulation of the classical depiction of the PP (Figures 1 and 2) commenced in 1931 with the discovery of glucose 6-phosphate dehydrogenase (Glc 6-P DH) by Otto Warburg, a German biochemist and a Nobel laureate of extraordinary distinction and influence. Warburg named the enzyme Zwischenferment (intermediate enzyme) in order to designate the branching of Glc 6-P away from the pathway of fermentation (glycolysis). In 1935 a second enzyme, 6-phosphogluconate dehydrogenase, was isolated together with a new pyridine nucleotide coenzyme (Wasserstoffiibertragendes: hydrogen-carrying cofer-ment), which is now called NADP+. Warburg recognized that NADP+ was chemically and functionally different from the NAD+ coenzyme of glycolysis. With these discoveries Warburg had opened the way into an alternate path of glucose dissimilation. Warburg believed that the NADPH product of these reactions was the substrate of cellular respiration and was thus a prime chemical source of aerobic cellular energy. This view was strongly held and disseminated for the next 20 years, such that the PP was first called the "direct oxidative pathway" in order to distinguish it from the path of fermentation of glucose and lactic acid formation in muscle.

The proposition linking NADPH and respiration (eqns. [1]-[3]) was shared by Erwin Haas, who was a member of Warburg's Berlin-Dahlem laboratory. (Haas fled Germany in 1938 and proceeded to the University of Chicago, where he was joined by Bernard Horecker, a fresh Ph.D. graduate, who was already exhibiting a flair for enzymology.) Haas possessed much of Warburg's data, understood his methods very well, and had a plan of research to test the proposed role of NADPH in respiration. Haas and Horecker set out to isolate a putative NADPH-cytochrome c reductase (eqn. [1]) in order to demonstrate the existence and nature of an ch2oh c

14ch2oh

ch2op

Hy/O

ch2op

Gra 3-P

ch2op

Gra 3-p

ch2op

chooh

ch2op

Ery 4-p

ch2op

ch2op

ch2op

ch2op

Fru 6-p

FIGURE 2 The proposed reaction sequence of the non-oxidative segment of the classical F-type pentose pathway in vitro. The [1-14C]-ribulose 5-P substrate is the labeled product of the Figure 1 reaction scheme and the reactions display the theoretical distributions of C-isotope from pentose 5-P into the carbon atoms of labeled intermediates and products. The enzymes catalyzing the reaction steps are: e, ribulose 5-phosphate 3-epimerase, i, ribose 5-phosphate isomerase; TK, transketolase; TA, transaldolase. The blue panels show the two-carbon active glycolaldehyde group that is conveyed by TK mass transfer and group exchange. The red panels show the three-carbon dihydroxyacetone group that is carried by TA mass transfer and exchange (see text and glossary for other detail).

enzyme that was hypothesized to be the "missing link" in a respiratory pathway between reduced pyridine nucleotide and oxygen via the cytochrome system (eqn. [3]):

NADPH + H+ + Cyt-cFe3+ NADPH cytochrome c reductase NADP+

2H + Cyt-cFe2+ + 0.5 O2 Cytochrome oxidase, H2O + Cyt-cFe3+ [2] Sum reaction:

The search for an enzyme activity was successful and a reasonably pure flavoprotein, NADPH-cytochrome c reductase, was isolated from yeast in 1940. Research was interrupted and not taken up again until the end of the

Second World War. By 1948, Horecker had returned to the project and soon extensively purified the enzyme from liver. However, by that time, there was growing evidence that the oxidation of NADPH was not the source of reducing equivalents for respiration and the entire proposal was abandoned in 1951, when Albert Lehninger showed that NADH was the "low-redox" respiratory chain substrate for oxidative phosphorylation in mitochondria.

The understandable disappointment with the realization that NADPH-cytochrome c reductase had no role in respiratory energy production (it is now known to be part of the cytochrome P-450 hydroxylation-detoxification scheme) directed Horecker's interest to a larger problem, namely, an inquiry into the nature, fate, and metabolic role of the pentose product of 6-Pg decarboxylation (Figure 1). It is also noteworthy that the British biochemist Frank Dickens (Courtauld Institute, UK) had been making pioneering investigations on this topic since 1936. However, a solution of the problem was made in the USA, where metabolic biochemistry and enzymology were flourishing in the 1950s. The dominant contributions came from the laboratories of Bernard Horecker, Ephraim Racker, and Seymour Cohen. Moreover, a further stimulus was the spectacular investigation made in Melvin Calvin's laboratory at UC Berkeley, which led to a Nobel prize and an understanding of the path of carbon fixation in photosynthesis (PS). Calvin's progress was heavily dependent on success in research by the aforementioned biochemists to resolve the enzymology and chemical problems posed by the reactions of Figure 2.

By 1950 all investigators possessed strong clues which served as signposts for an ultimate elucidation of a PP reaction scheme. (1) There was clear evidence that an alternate path of Glc 6-P dissimilation existed in yeast, red cells, and other animal tissues. (2) Dickens had confirmed that 6-Pg was oxidatively decarboxy-lated at carbon 1, to yield pentose 5-phosphate and other sugar phosphate products including a putative tetrose-P. He also demonstrated that Rib 5-P was oxidized at 5 times the rates of arabinose 5-phosphate (Ara 5-P) and xylose 5-P (theoretical products of 6-Pg decarboxylation). (3) Finally, as early as 1938, Zacharias Dische, using red cell lysates, found that inosine and inorganic phosphate (Pi) were converted to triose and hexose phosphates. This last important finding of the possible end products of Rib 5-P dissimilation was confirmed in 1946 by Schlenk and Waldvogel, who showed Glc 6-P formation from Rib 5-P using rat liver extracts.

Post Second World War Discoveries of Reactions in the Nonoxidative Segment of the Pentose Pathway

With the above background, between 1950 and 1955, very significant discoveries of enzyme and substrate reactivities followed, which were incorporated into a reaction scheme (mechanism) for the "classical" nonoxidative PP (Figure 2). The diagram in the figure is also designated the F-type (for fat-cell) PP, because it was later shown to uniquely measure a quantitatively large contribution to metabolism when Glc was converted to fatty acid and triglyceride in insulin-stimulated adipocytes. The findings may be summarized in the following temporal conjunction. In 1951 Cohen's group showed that Rib 5-P and Ara 5-P were formed from 6-Pg oxidation. Horecker and his collaborators confirmed Rib 5-P formation and unequivocally established that Ru 5-P was the first pentose-P formed from 6-Pg decarboxylation. They also identified a new enzyme, ribose 5-phosphate isomerase, which catalyzed the interconversion of the two pentose phosphates (see Figure 2). In 1952, Horecker and Smyrniotis, using a liver enzyme preparation, reported the important observation that Rib 5-P was metabolized to sedoheptulose 7-phosphate (Seh 7-P). This seven-carbon ketosugar ester was also found by Andy Benson, in Calvin's laboratory, and was identified as an early product of PS carbon fixation. Seh 7-P was formed by the action of transketolase (TK) (see Figure 2). TK was discovered by Racker and his collaborators in 1951, and it was demonstrated that it catalyzed the transfer of a two-carbon fragment (an active glycolaldehyde group) from appropriately structured ketulose-sugar donors to a wide selection of aldo-sugar acceptors. Two of its donor transfer actions, using different aldo acceptors, are shown as blue rectangular panels in Figure 2. The enzyme requires Mg2+ and thiamine pyrophosphate to be active and the list of 15 of its glycolaldehyde acceptor substrates is tabulated. Horecker's group discovered another broad-specificity group transferring enzyme, namely, transaldolase (TA), which catalyzed the reversible transfer of a dihydroxyacetone-enzyme-bound moiety (shown as red panels in Figure 2) from Seh 7-P to glyceraldehyde 3-phosphate (Gra 3-P), thereby forming Fru 6-P and a tetrose phosphate, which was neither isolated nor identified. The availability of synthetic erythrose 4-phosphate (Ery 4-P) enabled Kornberg and Racker to demonstrate the reversal of the TA reaction (eqn. [4]), thus satisfying the reason for its specific inclusion as an intermediate in the reaction scheme of Figure 2. Ery 4-P probably only exists in exceedingly low concentrations in any tissue and to date there is no evidence that it has ever been correctly measured in, or isolated from, any preparation carrying out PP metabolism.

Finally, in 1954, a third ketopentose ester, xlulose 5-phosphate (Xlu 5-P), was isolated as a product of Rib 5-P metabolism by Ashwell and Hickman. Racker showed that this new intermediate, rather than Ru 5-P, was a definitive substrate of TK. The ribulose 5-phosphate-3'-epimerase (see Figure 2) that catalyzed the formation of the ketosugar imparted the transconfiguration to the hydroxyl at C-3, which is the necessary stereochemical condition for substrate reactivity. The 30-epimerase was simultaneously purified in 1956 by Stumpf and Horecker at NIH and by Dickens and Williamson in the UK (Williamson later became the lifelong collaborator of Hans

Krebs) in the UK. In summary, the above research had uncovered an array of substrates and enzymes that could possibly satisfy the minimum requirements for a new pathway that connected the product of 6-Pg decarboxylation with the formation of hexose and triose phosphates.

Finding a Reaction Sequence for the Nonoxidative Pentose Pathway

It is possible to draw various theoretical schemes that oblige the arithmetic conjunction of five carbon sugars with a summary outcome of sugar products that contain six and three carbon atoms, respectively. That variety is greatly enhanced if reactions by aldolase (Ald) are included. Aldolase occupies the same cellular compartment as the PP; it is a dihydroxyacetone 3-phosphate (DHAP) group transferring enzyme, with a catalytic capacity that is usually much greater than TK or TA (a notable exception is adipose tissue where Ald activity is low and only approximates the activity of TK and TA). Ald also has a broad substrate array of aldo-sugar phosphate acceptors, most of which are the same substrates as those involved in TK and TA reactions. (It has never been clear why the pioneering investigators of the nonoxidative PP assigned aldolase a role of catalytic "silence.")

The results of the two experiments that aimed to identify the reaction sequence (mechanism) of the PP were published by the Horecker group in 1954. Briefly, Horecker adopted a prediction-labeling technique using [1-14C]- and [2,3-14C]-Rib 5-P as substrates and enzyme preparations from rat liver, pea leaf, and pea root tissues to catalyze the formation of 14C-labeled hexose 6-P. It was assumed that the position and degree of any 14C- labeling in Glc 6-P formed from these variously labeled substrates would indicate the nature and order of the reactions involved in Fru 6-P formation (see Figure 2). The enzyme preparations were made from acetone-dried powders of the above tissues. They were therefore free of all nucleotides and thus confined the reactions to a hexose 6-P end point by preventing any possibility of its recycled return through the oxidative segment, with consequent further scrambling of a "predicting" isotope-labeling pattern. Mg2+ was also omitted from the reaction mixture in order to inhibit the activity of fructose bisphosphatase and thus the formation of a contaminating Fru 6-P resulting from aldolase manufactured Fru 1,6-P2 using the triose-P products of TK-catalyzed reactions.

The experiments with liver enzyme preparation were of 17 h duration. Ribose 5-P was rapidly utilized during the initial 3 h and Seh 7-P also accumulated during this early period. Only with the slow decline in Seh 7-P at 6 h was there an increased linear production of Glc 6-P, which was harvested after 17 h and degraded carbon atom by carbon atom to produce the 14C distribution pattern of the whole molecule. The results, using [1-14C]-Rib 5-P as substrate showed that the Glc 6-P product was labeled with 14C isotope in carbons 1 and 3 (see Figure 2) with a C-1/C-3 ratio of 3 (74% of the 14C isotope in C-1 and 24% in C-3). Horecker "tentatively" proposed that the reaction scheme of Figure 2 was consistent with the above isotope distribution. Clearly the consistency is not there, because the pooled [1,3-14C]-Fru 6-P formed in the TA and second TK-catalyzed reactions in Figure 2 impart a 14C content in C-1 and C-3 with a ratio of 2 (twice as much in C-1 as C-3). The difference is serious and the isotope distributions cannot be reconciled with the Figure 2 reaction scheme, nor can the difference be attributed to the tedious analytical and degradative procedures, since the percentage errors in the determination of C-1 are only 2%; C-2, 2.7%; and C-3, 1% with a cumulative percentage error of 12% for the estimate of all carbons of the molecule. The results of the companion experiment conducted by Gibbs and Horecker in1954 used [2,3-14C]-Rib 5-P as substrate and liver, pea root, and pea leaf enzyme preparations made from the acetone-dried powders of these tissues. The results deviated even more radically from the predictions imposed by the sequences of Figure 2 than the above data using [1-14C]-Rib 5-P. It is emphasized that in the publications of these two studies, an ordered series of chemical equations for the nonoxidative PP was only "tentatively" proposed and a metabolic map was not shown. However, in 1955, Horecker authored two important and substantial reviews of carbohydrate metabolism and presented for the first time the diagram illustrating the new metabolic pathway (still with the "tentative" caveat). This illustration (Figure 2) is still the chart of the PP or pentose cycle (PC)—a pathway that has featured in textbooks of general biochemistry since 1956. It is astonishing that such a profound disagreement between practice and theory was so uncritically ignored by the general community of biochemists and glossed over by the very few who drew it to attention in the review literature. This indifference resulted in the prompt inclusion of the scheme of Figure 2 into the canon of metabolic biochemistry without further inquiry. It is also ironic that the participation of a large number of PP reactions into the path of carbon in PS added confidence and prestige to the status of the PP. However, there were two other comprehensive investigations of the mechanism of the PP, one by Joseph Katz and co-workers in 1955 using [1-14C]-Rib metabolism in liver slices and the other by Howard Hiatt in 1957 using the same labeled substrate in mouse liver in vivo. These workers did not find 14C distributions in the labeled glucose product with twice as much isotope in C-1 as C-3, but instead found the carbons equally labeled. These independent early failures to confirm the predictions of Figure 2 were also ignored.

Other than valuable research by Patricia McLean and her collaborators at the Courtauld Institute (UK), who investigated the enzymology, endocrinology, and occurrence of PP reactions in many animal tissues, all fundamental research on the mechanism of the pathway essentially ceased in 1957 and was not taken up again for another decade. Instead the era of the quantitative measurement of pathways of carbohydrate metabolism had dawned and PP measurements featured hugely. This emphasis on quantitation is best summarized in the following statement by Harland G. Wood in 1955. "The determination of the relative role of different pathways in normal living cells is without doubt of the greatest fundamental importance to our understanding of life processes and will in the future require more attention in all fields of metabolism." Wood (Western Reserve University Medical School, Ohio) collaborated with Joseph Katz (Cedars of Lebanon Hospital, Los Angeles) over the next eight years in the development and elaborations of measurement theory for an entity denoted by H. G. Wood and later defined by Wood and Katz as the pentose cycle (PC).

The Quantitation of the Pentose Cycle: Theory and Practice

Between 1958 and 1979 a dozen elegant theoretical papers were published that provided the mathematical basis and formulas for measuring the F-type PC using 14C-labeled substrates. All of these methods depended on a quantitative solution of the problem posed by the recycling of 14C isotope distributions that emerge by the metabolism of the substrates [2-14C]- or [3-14 C]-glucose in PC. Calculating the different distributions of labeled carbon to infinite cycles, for all percentage contributions of PC, is a difficult mathematical problem, which was solved by Joe Katz. Katz is not only a gifted biochemist but also an equally talented mathematician and innovative metabolic theorist. The acceptance of a PC definition imposed agreement that all 14C-labeled Fru 6-P formed by the Figure 2 reaction sequence is converted to Glc 6-P and recycled again through the oxidative segment reactions. The quintessence of all measurement methods has involved the development of mathematical expressions describing the rhythmical and ordered redistributions of either carbon 2 or 3 from labeled substrate glucose, into positions 1, 2, and 3 of the hexose 6-P products for any percentage contribution of PC. Such a theoretical distribution is a unique property of the PC. Experimental data for the C-1/C-2 and C-3/C-2 ratios, that have definite limits and values imposed by Katz and Wood theory, measure the degree of redistribution of 14C from the above labeled substrates into the top three carbons of the Glc 6-P product, which is isolated and analyzed for 14C distribution following labeled substrate metabolism. The ratios, in their appropriate measurement equations, express the PC contribution relative to the total metabolism of glucose. The above statements cannot be qualified. They derive from the reaction sequence of Figure 2, which is the mechanistic basis for all the formulas of all the "measurement" papers. The precise values for C-1 and C-3 and the ratio of their isotopic labeling are the "identity" badge of the classically defined PC reaction sequence, which is the foundation of its theories of existence and quantitation. It was therefore intriguing to note, that notwithstanding the initial ambiguities in the establishment of the Figure 2 scheme and the failed efforts of the Katz and Hiatt experiments to support it, that no less than ten independent efforts, over nearly 30 years, have failed to find any significant level of PC in liver using the Katz and Wood method. Liver is a rich source of the enzymes of the PP and it can provide inter alia a ready display of the reactions of Figures 1 and 2. Thus, the failure by all measurement investigations to find an F-type PC in liver was mystifying.

The Search for a New Reaction Scheme for the Pentose Pathway

A solution to the mystery was sought in the author's laboratory, with investigations that began in 1965 at the University of New South Wales and later at the Australian National University. Work commenced with the propositions that the scheme of Figure 2 may be an erroneous interpretation of the PP and the coupling of Figures 1 and 2 reactions into a metabolic cycle (PC) was possibly ill-conceived. The following three sets offindings summarize selected aspects of progress in the unraveling of a new PP reaction sequence in liver that does significantly contribute (20-30%) to glucose metabolism.

First, the original experiment performed by Horecker in 1954 was repeated using [1-14C]-Rib 5-P and exactly the same preparation of liver enzymes. However, reaction mixtures were sampled for the labeled Glc 6-P product at a series of much shorter time intervals and right up to the 17 h termination point described in the original work. The results showed a patterned assortment of label distributions in Glc 6-P, that drifted from 8 to 17 h towards the isotope composition in C-1 and C-3 that was originally reported by Horecker. Notably in this study, the C-1/C-3 ratio at 17 h, was the much-sought value of 2. Moreover, in the seven-time samples, which commenced at 1 min, Glc 6-P was heavily labeled in C-2, C-4, and C-6, while C-1 and C-3 only began to accumulate 14C-isotope after 3 h of reaction. Although this was a study in vitro, it is obvious that liver cells in vivo do not take between 3 and 17 h to elaborate a path of metabolism and that more enlightening events were being revealed by the isotope distributions in the samples analyzed between 1 and 30 min of reaction. A carbon balance analysis of all compounds in the various reaction mixtures showed that the intermediates of Figure 2 only accounted for 80% of the carbon in the Rib 5-P substrate. The compounds comprising the missing 20% were identified as sugar phosphates, mostly ketuloses (see Figure 3). They were isolated, identified, and shown to be radioactive. These keto-ester sugars were Seh 1,7-P2, D-manno-Heptulose 7-P; D-glycero-D-altro-octulose 1,8-P2(D-g-D-a-Oct); D-gly-cero-D-ido-octulose 1,8-P2(D-g-D-i-Oct); and a small amount of Ara 5-P (see Figure 3). Octulose (Oct)-, mono- and bisphosphates, and Seh 1,7-P2 were also isolated and measured in fresh liver. Figure 4 shows the structures and reactions of these sugar esters in a new and much modified reaction scheme for the PP in liver. The new intermediary compounds were easily isolated from all incubations from 30 min to 17 h. The scheme of Figure 4 shows the new PP with prediction 14C-labeling patterns in the intermediates and products of the reactions. The reaction scheme for Figure 4 was initially formulated from the distributions of 14C in the labeled Glc 6-P and D-g-D-i-Oct 1,8-P2 formed from [1-14C]-Rib 5-P during the early intervals of the repeat experiment performed by Horecker and co-workers in 1954. The new pathway, called L (liver)-type PP, is distinguished from depictions of the classical F-type PP by the inclusions of Seh-1,7-P2 and octulose (Oct)-, mono-, and bisphosphates together with Ara 5-P as new intermediates. Aldolase, phosphotransferase (PT), and arabinosephosphate isomerase are new enzymes. Mass transfer catalysis by TA was omitted, but the effects of active TA-exchange reactions (TAX), which accounted for the 4,6-14C labeling of Glc 6-P, were encountered in all samples. The second TK reaction forming hexose 6-P products in the L-type PP (Figure 4) used D-g-D-i-Oct 8-P as a substrate and TK was also found to catalyze very active exchange reactions as does aldolase. A clear demonstration that aldolase is a mandatory catalyst in liver PP involved the immuno-chemical evidence of Bleakly and co-workers in 1984, who showed the cessation of all hexose 6-P formation when liver aldolase antibody titrated the removal of aldolase from the system where Rib 5-P was reacted with the rat liver enzyme preparation that was used to establish the scheme in Figure 2. Irrespective of the other contrary data, this evidence alone showed that there was another reaction mechanism involving Ald in liver PP. The claim that aldolase is an essential enzyme in the PP was also supported by data of Cori and Racker, who, in an in vitro "construction" of a PP preparation for the complete oxidation of Glc, noted the formation of Oct-P and the need to include aldolase and sedoheptulose 1,7-bisphosphatase for the construction system to work.

FIGURE 3 Gas liquid chromatogram (GLC) of the dephosphorylated derivatized sugars formed after 30 min reaction of rat liver enzyme preparation with ribose 5-phosphate. The procedures for sample processing and GLC are given in Williams, J. F., Clark, M. G., Arora, K. K., and Reichstein, I. C. (1984) Glucose 6-phosphate formation by L-type pentose pathway reactions of rat liver in vitro: Further evidence. Seyler's Zeit. Physiol. Chem. 365,1425-1434.

FIGURE 3 Gas liquid chromatogram (GLC) of the dephosphorylated derivatized sugars formed after 30 min reaction of rat liver enzyme preparation with ribose 5-phosphate. The procedures for sample processing and GLC are given in Williams, J. F., Clark, M. G., Arora, K. K., and Reichstein, I. C. (1984) Glucose 6-phosphate formation by L-type pentose pathway reactions of rat liver in vitro: Further evidence. Seyler's Zeit. Physiol. Chem. 365,1425-1434.

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  • josh duncan
    Who discovered pentose phosphate pathway?
    2 years ago
  • otho
    Who discover pentose phosphate pathway?
    2 years ago
  • ray
    Who discovered hexose monophosphate pathway?
    2 years ago

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