F. Gabrielli Universitv of Pisa Pisa. Italy
I I
Gluconeogenesis: A Teaching Pathway Criteria to establish a metabolic pathway
Gluconeogenesis is the metabolic pathway that synthesizes elucose from ovruvate. This uathwav is hioloaicallv .- . very important because in this way non-carbohydrate comvounds can he converted to elucose (1-4). Gluconeo~enesis has heen studied extensiveliin mammalian liver and it has been shown that the gluconeogenic glucose produced in this organ is essential for the life of the whole organism. In conditions of prolonged fasting, low carbohydrate diet or diabetes, nerves and blood cells which utilize primarily glucose as a source of energy, can not survive without a continuous supply of glucose produced by gluconeogenesis in the liver and kidney (3). The reverse of the glycolytic process-gluconeogenesiswas clearly recognized by Cori and Cori as early as 1929 ((21, see reference therein) but more than 30 years were necessary to reach a clear understanding of the whole pathway. During these years several schemes for the gluconeogenetic pathway were proposed and replaced with different ones as the experimental work proceeded, until the last scheme, which incorporates all the experimental results in a uniaue wav. As the studv of eluconeoaenesis proceeded, criterL for Goper experikentaf and for a correct evaluation of the data were developed. These criteria are of genera! meaning, and can he usefully applied to evaluate the validitv of other vathwavs and to direct the research for new pathways.- In this paper, the different schemes of gluconeogenesis, proposed at subsequent times during the elucidation of this pathway in mammalian and avian liver, will he reviewed. This may help in understanding how the criteria to be discussed have heen developed, and on which experimental data these criteria are hased. The first scheme to he proposed for a gluconeogenetic pathway suggested that the synthesis of glucose from pyruvate occurs by direct reversion of glycolysis (1, 2). Since all the reactions of glycolysis are shown to he reversible in uitro, the possibility of glucose synthesis in uiuo by this reaction was considered ((1.2) see also reference therein). Suhsequently, several experimental data indicated that alternative reactions catalyzed by enzymes not involved in glycolysis could participate in the synthesis of glucose from non-eluconeoeenic comuounds. The discoverv in liver cells of a specific gucose-6-dhosphate phosphatase and a specific fructose-1,6-diphosphate phosphatase suggests that these two reactions may participate in glucose synthesis from fructose-1,6-diphosphate( ( I ) , see reference therein). This proposed pathway has as intermediates the same exose vhosuhates of the alycolytic pathway, but utilizes two . . different enzymes. ~ u r & r eqe;imentsproved that the two specific phosphatases are key enzymes of gluconeogenesis, and that in uiuo the corresponding glycolytic reaction can not participate in the synthesis of glucose because it is thermodynamically feasible only in the direction of glucose degradation (1). Another reaction that earlier was thought to he irreversible in uiuo is the phosphorylation of pyruvate (1,5). In order to overcome this prohlem, alternative pathways for the synthesis of P E P (phosphoenolpyruvate) from pyruvate were proposed. The existence of these alternative
-
I am extremely grateful to Professor C. Baglioni for having read and corrected the manuscript. The work was supported by grant No. CT 75.00525.04. of Italian C.N.R. 86 / Journal of Chemical Education
GLYCOGEN
t t
GLUCOSE
t t t
PHOSPHOENOLPYRUVATE + CO,
PHOSPHOENOL-OXALOACETATE
FUMARATE
-
MALATE
11
ACETYLPHOSPHATE + CO.
LACTATE _ 2 PYRUVATE ~
+co,
OXALOACETATE
Figure 1. Scheme of carbohydrate synthesis from lactate and pyruvate according to Lipmann, 1941 (8)and (double line arrows)according to Solomon el al., 1941 (9).in the Lipmann scheme two acatylphosphate are necessary to produce one fumarate.Phosphoenol-oxaloacetate = CH-COOH
II
C-COOH
pathways is supported by some experiments that can not he explained by a direct phosphorylation of pyruvate to P E P via the pyruvate kinase reaction. I t has been shown that kidney cortex extracts catalyze the conversion of fumarate and malate to P E P (6) and that less than 2% of lactate carhoxyl carhon is converted to liver glycogen (7, 8). These data suggested that only carhon 2 and 3 of the lactate molecule participate to P E P synthesis from pyruvate and that dicarboxylic acids are metaholic intermediates. In 1941 Lipmann (9) proposed that the synthesis of gluconeogenetic P E P is initiated hy decarhoxylation and phosphorylation of pyruvic acid with formation of the two carbon unit acetyl phosphate; two acetyl phosphate molecules condensate to form fumarate; the dicarhoxylic acid is phosphorylated to give phosphoenol-oxaloacetate and then the phosphodicarboxylic acid is decarboxylated to form phosphoenolpyruvate (PEP) (Fig. 1). A short time later, Solomon et al. (9) by feeding rats with lactate and [14C] bicarbonate, established that the isotopic carhon is incorporated into glycogen; these authors suggested that COz is a gluconeogenic metabolite and proposed a pathway for the synthesis of P E P from pyruvate with a new reaction for the synthesis of the dicarhoxylic acid. Oxaloacetate is synthesized by direct carhoxylation of
pyruvate, converted to malate and then to fumarate; PEP is synthesized from fumarate through the reactions proposed by Lipmann (Fig. 1).Solomon et al: (9) point out that, because of the symmetry of the fumarate molecule, half of the labelled CO? incorporated into pyruvate is lost in the decarboxylation of fumarate and the other half is incorporated into glycogen. Topper and Hastings (10) using [14C] COz, [2-14C] pyruvate and [I-14C]acetate and looking at the position of the isotopic carhons incorporated into glucose units of glycogen (synthesized in vitro by rahbit liver slices) were ahle to establish that about 80% of the pyruvate molecules are randomized before being incorporated into these glucose units. These authors indicated that the carboxylation of pyruvate to form oxaloacetate and equilibration of oxaloacetate with symmetrical dicarboxylic acids (dicarboxylic acid shuttle) occurs four times as fast as the direct phosphorylation of the pyruvate. Topper and Hastings (10) point out that their isotopic experiments can not establish "whether phosphorylation of dicarhoxylic acid can or does occur." They believe that it is not necessary to postulate the formation of phosphoenolmalate orland phosphoenoloxaloacetate as intermediates for the PEP synthesis, because Lardy and Zigler (11)had shown that it is experimentally possible to synthesize PEP by direct phosphorylation of pvruvate using ATP and. as catalvst. kinase. . . . .Dvruvate -. The experiments of ' ~ o ~ and ~ e Hastings r (10) plus Lardy and Zigler (11) can be explained assuming that 80% of the pyruv& molecules enter in the dicarh~xylicacid shuttle before being phosphorylated and that the remaining 20% are incorporated into glucose without any change or loss of carbon atoms via direct phosphorylation (Fig. 2). In this scheme of gluconeogenesis, dicarhoxylic acids and COz do not have a role in the synthesis of PEP even if isotopic carbons of COz could be incorporated in the glucose unit of glycogen because of the shuttle. GLYCOGEN
t t
to eive PEP and COa. . (13). . The new scheme is orooosed on the- basis of known enzyme activities and of &e&bolic intermediates known to be normal constituents of the cell. However, this proposed pathway is thermodynamically unfavorable in the direction of phosphoenolpyruvate synthesis, not less unfavorahle than the direct reversion of the pyruvate kinase reaction (I). Utter and Kurahashi (11)and Krehs (I) indicate that a system of enzymatic reactions capable of maintaining a high NADPH/NADPC ratio in the cell would make energetically possible the PEP synthesis via the proposed pathway. Proofs of this possibility were given by Ochoa and coworkers (12). The proposed pathway bas no fumarate among its intermediates, hut a rapid equilibrium between malate and fumarate can be postulated. However, even if the new pathway seemed to give a logical explanation to all the experiments it was found incorrect after a few years. Mendicino and Utter (2, 15) found that isolated chicken liver mitochondria that lack nvruvate kiuase as well as malic enzyme, are able to synigesize PEP from pyruvate. This sueeests that in these mitochondria a oathwav for the PEP sy%hesis is present different from thk one proposed. In fact Utter and Keech (6) were ahle to detect in the chicken liver mitochondria a new COz-fixing biotine enzyme (16): the pyruvate carboxylase that catalyzes the carboxylation of pyruvate to oxaloacetate using ATP as energy donor. The discovery of the mitochondrial pyruvate carboxylase solves the problem of PEP synthesis in gluconeogenesis; Utter and Keech (18) proposed that pyruvate is directly converted into oxaloacetate by the pyruvate carboxylase reaction and oxaloacetate into PEP hy the already known phosphoenolpyruvate carboxykinase (Fig. 4). This pathway is the simplest mechanism for PEP synthesis among the ones previously proposed. It requires the breakage of two high energy bonds, and it makes the PEP synthesis from pyruvate thermodynamically feasible. In chicken liver, pyruvate carhoxylase and PEP COz kinase are both mitochondrial and the other enzymes necessary for the gluconeogen-
GLUCOSE
t t t t
-
PHOSPHOENOLPYRUVATE
..
PYRUVATE
+co,
OXALOACETATE
MALATE FUMARATE Figure 2. Scheme of carbohydrate synlbsis from pyrwate acwrding to Topper and Hastings, 1949 (10).
As suggested by Topper and Hastinge (lo), the problem of PEP synthesis from pyruvate can not he solved hy the isotopic techniques above indicated. In fact, only investigations directed at identifying the enzymes involved in the first step of gluconeogenesis will resolve this problem. In 1954 Utter and Kurahashi (11) and Krehs ( I ) proposed a new scheme for the PEP synthesis from pyruvate (Fig. 3). Pyruvate is converted to malate, malate to oxaloacetate, and oxaloacetate to PEP. This new scheme is in agreement with the known isotopic experiments and is supported by the discovery of the two enzymatic activities: malic enzyme, that catalyzes the carboxylation of pyruvate to malate using NADP as coenzyme (12). and phosphoenolpyruvate carboxykinase, that catalyzes the decarboxylation and phosphorylation of oxaloacetate by ITP (or GTP)
Figwe 3. Scheme 6f glucose synhsis hom lactate and pyrvvate according to M e r and Kurahashi. 1954 (11)and accoding to Krebs. 1954 ( 1 ) .
Volume 53.Number 2, February 1976 / 87
Figure 4. Scheme of glucose syntbis from pyrwate. The phosphoenolpyrwate synthesis from pyruvate is acmding to Uner and Keech, 1963 (17). The other Dans were from Krebs (3).
esis are localized in the cytoplasm. Therefore, during the gluconeogenesis P E P is synthesized inside the mitochondrion, diffuses in the cytoplasm, and in that compartment is converted to glucose (19). This pathway has been found valid for those liver cells in which pyruvate carhoxylase and P E P Con kinase are both localized in mitochondria. However, in liver of animals like rat, mouse, and hamster, pyruvate carboxylase is mitochondrial and P E P C02 kinase cytoplasmatic (20). Lardy and his school (21-23) have shown that a more complex pathway for P E P synthesis from pyruvate is then present (Fie. 5). In the livers of these animals, oxaloacetate synthe&d in the mitochondria can not diffuse in the cytoplasm because the mitochondria1 membrane is impermeable to this compound. Inside the mitochondria, oialoacetate is converted to malate or aspartate and these compounds diffuse into the cytoplasm where they are converted again to oxaloacetate. The conversion of oxaloacetate to malate and the diffusion of this dicarhoxylic acid into the cytoplasm has the double function of transporting outside the mitochondria oxaloacetate and "reducing power" (NADH) that can be utilized a t the level of the phosphoglyceraldehyde reaction. Aspartate is carrier of the sole oxaloacetate for the gluconeogenic pathway and it is utilized when the NADH for the eluconeoeenesis is svnthesized in the cvtoplasm. Other experiments prove that malic enzyme is not a gluconeogenetic enzyme but is primarily involved in liponeogenesis. Malic enzyme activity decreases in the liver of fasting rats, and increases when the animal is refed with a high carbohydrate diet (24). Furthermore, this enzyme has a high value of K , for NaHC03 (2). These data indicate that the malic enzyme works in the direction of pyruvate, Con, NADPH synthesis, and they also confirm indirectly that pyruvate carhoxylase is the COz fixing enzyme responsihle for dicarboxylic acid synthesis for gluconeogenesis. This history of the experiments performed t o elucidate the simple steps of gluconeogenesis is a good example of how advances in our understanding of biochemical pathways are
-
88 / Journal of Chemical Education
Figure 5. Scheme of glucose synthesis according lo Lardy, 1965 (21).
Figure 6.
E = Enzyme: A. B. C. D, and X = Metatolio intermediates.
obtained. Furthermore, these experiments point out the loeic of the metaholic pathway scheme and show also the techniques and criteria necessary to demonstrate a metabolic pathway at the molecular levcl. Criteria and Experimental Procedure to Establish a Metabolic Pathway
The structure of a metaholic pathway is very simple; it consists of a group of enzymes working in sequence in the sense that the product of the first enzvmatic reaction is substrate of thesecond and so on (Fig. 6): Each metaholic intermediate mav be a reaeent for several different chemical reactions. ~ o b e v e rit, Gill participate a t a verv. high - rate onlv in the reaction that is catalvzed hv a specific enzyme; even a weak covalent bond is rarely hroken by thermal motion within the cell, and reactions concerned with formation or breakage of covalent bonds are catalyzed bv enzyme (36). Each specific catalyst, increasing the ratd of the relative reaction, will contribute to the synthesis of the last product. Substrate specificity combined with the catalyzing power of the enzyme will make possible the rapid transformation of compound A into E with a rate consistent with the cell needs. In order t o prove the existence of a metaholic pathway it is necessary to demonstrate that all the enzymatic activities catalyzing the sequence of reactions and all the metaholic intermediates are nresent in a cell. These are necessary but not sufficient ckditions in order to demonstrate a metaholic nathwav. ". hut additional experimental proofs are required. I present here what I consider significant experimental proofs that must accompany the necessary conditions
+
(presence in the cell of the enzymes metabolites) in order to demonstrate a metabolic pathway. None of these proofs alone is sufficient to establish the existence of a metabolic pathway; however, these proofs reveal if a pathway is present and if i t can work in a cell. Free Energy Change of the Metabolic Pathway
The free energy change of the metabolic pathway provides the first indication that a pathway is thermodynamically feasible. I t can be calculated adding the free energy change of each reaction in the pathway (1,2,11,25-27). A preliminary indication of the energetic behavior of a pathway is obtained using free energy change in standard conditions or for a unique concentration value of all reagents and products of each enzymatic reaction in the pathway, and using pH, temperature, 02, and COz pressure values close to the physiological ones (1,2,11,25-27). This tvoe of calculation has been used bv Krebs (1.3) to postulat;?'that the exokinase, phosphof&ctokina&, A d ovruvate kinase reactions are irreversible. However. the z a n g e s of free energy calculated with unphysiologicalconcentrations of metabolites mav eive incorrect answers. A " typical example is the lactate dehydrogenase reaction (lactate NAD+ e Dvruvate NADH + H f ) that has a AGO = -15.54 kcal a n i a AG' = -6 kcal in the sense of pyruvate formation (AG' has been calculated with reagents and products in the concentrations of 0.01 mM a t 25°C and p H = 7). On the basis of this AG' value, the reaction in the cell should go only towards pyruvate NADH + H+ synthesis. However, i t has been proved by metabolic studies that in the liver cells this reaction can proceed in the opposite direction (3). I have calculated the AG of lactic dehydrogenase by the formula
-
+
+
+
AG
=
AGO + RTlog. [Pyruvate] [NADH] [H+] [Lactate] [NADf]
R is the gas constant, T is the absolute temperature, AGO value was taken from Burton and Krebs (25), the pyruvate and lactate concentrations and NAD+/NADH ratio were those determined in the rat liver cell (28, 29), and the H+ M. concentration was In this condition AG of the reaction in the direction of lactate formation is verv close t o zero (de~endineon the reagent and product concentrations found in the cells, AG varies between +0.34 and -0.95 kcallmole). Similar data have been obtained for the free energy change of the lactic dehydrogenase erythrocytes (38). These data indicate that the lactate dehydrogenase reaction is very close to thermodynamic equilibrium; hence, small changes in the concentration of the reagents andlor products c d a h i f t the reaction from one direction to the opposite. During active gluconeogenesis from lactate in liver cells, the reagents and products have t o be maintained a t certain steady concentrations that make thermodynamically possible the synthesis of pyruvate from lactate. Free enerev change calculations of each sten of ervthrocyte glycoly& (using physiological concentr&ions of the reagents and products) show that the maioritv of the metiholL reactions have negative AG (14). he ;her reactions have a very low, positive AG. The positive values of AG can be due to the technical difficulty of determining the actual content of the metabolite in the cell. I have repeated these calculations using the concentration of the glycolytic intermediates in the liver cells, and I found very similar results. These data show that three glycolytic reactions namely exokinase, phosphofructokinase, and pyruvate kinase have strong negative AG;all other reactions have AG very close to zero. The strone neeative value of the free-enerw variation of the erythrocse & hepatocyte glycolysis is prymarily due to the AG values of these reactions; there are reactions unique
to the glycolysis and irreversible in the cell. The other reactions in common with the glycolysis and gluconeogenesis have AG values verv close to zero and thev can oroceed in both directions. The AG (calculated for the actual concentrations of reagents and products) of each reaction in the pathway has t o be negative; if one reaction has nositive AG. even if the total ZG of the pathway is negativi, it indicates that the reaction is not in the pathway. The validity of the AG calculation is limited by the difficulty of determining the actual concentrations of reagents and products in the site where the reaction takes place in the cell. For thermodynamic reasons, in the cell the synthetic and the catabolic pathways of the same compounds are necessarily different. They may have some common reactions, but they necessarily differ for those reactions that have strong negative values of free-energy change. This is true for the svnthesis and deeradation of small molecular weieht " compounds (glucose, fatty acid, etc.) as well as for macromolecules (glycogen, proteins, RNA, DNA). Propertiesof the Enzyme
The enzymes that belong to a metabolic pathway show properties that are in harmony with the physiology of the pathway. Therefore, these properties can be used to verify whether or not an enzyme belongs to a pathway. Enzyme Specific Activity
The catalytic activity per g of fresh tissue (or per organ) of each enzyme in the metabolic pathway has to be equal to or higher than the metaholic rate of the whole pathway determined in uiuo as the rate of synthesis of the last metabolite (4). If an enzyme shows a specific catalytic activity lower than the rate of the pathway, this indicates that the enzyme can not be in the pathway. However, this criterion has to be used keeping in mind that i t is very difficult to estimate the real in uiuo catalytic activity of an enzyme. Furthermore. when the enzvme assav " is nerformed in crude preparations, other enzymatic reactions may interfere with it. On the other hand.. . purification causes losses of the enzyme (2,4). &
Variations of the Enzyme Catalytic Activity
When the metabolic rate of a whole pathway can be changed by altering the metabolic or hormonal conditions of the organism, the enzymes of the pathway should change their activity accordingly. When the catalytic activity of an enzvme decreases while the metabolic flow of the whole pathway increases, or vice versa, there is an indication that the enzvme is not in the nathwav (4.24). when the enzyme activity remains constant in value and the metabolic flow of the whole oathwav increases or decreases, no indication is obtained as to whether an enzyme belongs t o the pathway. The enzyme activity may remain constant because i t is high enough to support the increase in metabolic rate of the pathway, or i t does not decrease in the in uitro assay because the p&miological inhibitor or activator that operates in uiuo is lost or diluted during the isolation of the enzyme (2). Kinetic Properties of the Enzyme
The K , values for the substrates of an enzvmatic reaction assayed in both directions of a reaction, g&e useful indications for establishing which are the ~hvsioloeicalsubstrates of the reaction and from that, which>s thedirection of the reaction in the cell (2). The physiological substrates of an enzymatic reaction should have K , values related to their concentrations in the cell from which the enzyme is extracted. When the K , value of the suhstrate of an enzymatic reaction is much higher than the molarity of the substrate in the cell, this strongly suggests that it cannot be the substrate but is the product of the reaction. By this approach i t is possible to Volume 53, Number 2, February 1976 / 89
establish in which direction an enzymatic reaction proceeds in the cell. However. this criterion has to be used carefullv because kinetic experiments are made in conditions very different from the physiological ones (see previous paragraph); furthermore, i t is very difficult t o determine the actual concentrations of metaholites in the cellular compartment in which the enzyme is localized, particularly in the case of a metabolite not uniformlv distributed in the cell. The definition of the direction-of a reaction can be a key problem for the definition of a metabolic pathway, as has been seen for malic enzyme in the gluconeogenic pathway where no definitive information was obtained on the basis of free-energy changes (2). Other kinetic data may help to establish whether an enzvme belones to a metabolic ~ a t h w a vThe . effect of cellular metaholites as activators or inhibitors of the enzyme should be in harmony with the physiology of the pathway in which the enzyme is enclosed; e.g., inhibitory effects of Dhos~hataseand its AMP on the fructose-1.6-di~hos~hate activatory effect on the f r ~ ~ t o s e : b ~ h o ~ ~ kinase h a t eare in agreement with the concept that a high AMP level-associated with a low ATP level-stimulates glycolysis and inhibits gluconeogenesis and that fructose-1.6-di~hosphate phosphatase i s a gluconeogenetic enzyme and fructose-6phosphate kinase a glycolytic one (3,41. Specific inhibitors of the Enzyme
The use of specific inhibitors is a good test t o verify whether or not the enzvme belonrrs to a ~ a t h w a v The . administration in uiuo of compound, proved to be a specific inhibitor of an enzyme, must block the pathway a t the level of the reaction catalyzed by the inhibited enzyme (30, 31). As a consequence of this, the substrate(s) of the enzyme as well as its metabolic precursors will increase in concentration, whereas the enzyme product(s) and its metabolic derivatives will decrease in concentration. By this technique, called cross over-type plot, i t is clearly determined whether the inhihited enzyme belongs to a pathway; furthermore, the concentration changes of the cellular metabolites may provide information on other enzymatic reactions involved in the pathway.
a
Subcellular Locallzatlon of the Enzyme
When all the enzymes of the metabolic pathway are localized in the same cellular compartment, the product(s) of an enzymatic reaction will reach the enzyme that catalyzes the next reaction by free diffusion in the cell cytoplasm. When some of the enzymes of a metabolic pathway are localized in a subcellular compartment limited by a membrane and the remainine enzvmes are localized outside this membrane in another subcellular compartment, it is necessary to demonstrate that the metaholite that is synthesized in the first compartment and utilized in the second one can diffuse across the interposed membrane. If the membrane is impermeable to this metabolite the pathway is incorrect or incomplete. We have seen that in the rat the oxaloacetate which cannot diffuse through the mitochondrial membrane is converted to other metabolites which diffuse outside the mitochondria. These metabolites are converted again into oxaloacetate in the cytoplasm (21-23). In the rat the gluconeogenic pathway has two extra reactions with respect to the gluconeogenesis of other animals that have pyruvate carboxylase and phosphoenolpyruvate carboxykinase both localized in mitochondria.
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Use of Synthetic Substrates
Synthetic substrates can be added to biological systems (whole animals. ~ e r f u s e doreans. cell suspensions. tissue homogenates) in order to verify ifsnd how they are "tilized by the pathway that is under study. This approach is very powerful in the study of metabolism when metabolites containing isotopic atoms are used. The chemical properties of all the isotopes of an element 90 /
Journal of Chemical Education
are essentially the same, and molecules containing different isotopic atoms are metaholized bv the cell in the same way. Thiuse of isotopes thus allows study of the metaholic fate of compounds as well as the rate of their utilization in the cell. his experimental approach is particularly useful when a metabolic pathway is completely unknown. By this technique many p;oblemi of met&olisk have been solved and new pathways have heen discovered. We have discussed how with the use of isotopic carbon-carbonic anhydride i t has been established that COz fixation occurs in the eluconeoeenic nathwav (9). ~ k use e o f a compound: &iformly labelled in its carbon atoms oermits one to detect all the metabolic intermediates derivaied from the carbons of its skeleton. A more precise study can be made using the compound specifically labelled in single carbons. In this way it is possible t o follow the metaholic route of each carbon of the molecule and find out if the metabolic pathway is linear, branched, or a cycle. Using the pulse-chase technique i t is possible to determine which is the sequence of metabolites derived from the labelled precursors, in the order in which they are synthesized in the cell (35).In this technique a labelled precursor is administered t o a biological system and all the labelled metaholites present in the biological system are extracted and identified. The extraction of the labelled metabolites is carried out soon after the administration of the precursors and is repeated after short time intervals, sometimes after administration cf unlabelled orecursors (chase). The total isotopic content of the labelled metaboli& decreases with time, while the metaholites synthesized from it become labelled. The product of the first reaction that utilizes the labelled Drecursors, is the first t o become labelled and the first toieach a maximum of isotopic content; the other metabolites show similar behavior and by the time they become labelled and reach the maximal isotopic content, it is possible to determine the order in which they are synthesized. If the last metabolite is a final stable product, it increases constantly in isotopic content until all the radioactivitv of the nrecursor is contained in it. isotopes for metabolic studies may give erro~ k use e neous information when the transfer of isotooic atoms from one molecule to another occurs without a net conversion (32). This phenomena can take place by two different mechanisms 1) Erchanee reaction. It results from the oarticination of the malpcule containing isotopic atoms to a revemihle chemical system. In the cell this rystem can he an enzymatic reaetmn at equilibrium or an enzymatic.half reaction; in the first case the molecule containing isotopic atoms (substrate) is converted to product by a complete reaction and, because the reaction is at equilibrium, an equal amount of product present in the cell is reconverted to substrate. This oscillation permits the formation of labelled products without any net synthesis. In the second case, the molecule containing isotopic atoms participates to an enzymatic half-reaction. In this reaction an enzyme-substrate complex is formed but because of the absence of the other substrate(~)necessary for the complete reaction, the complex is hydrolyzed. However, the labelled product bound to the enzyme can exchange with a cold one added to the reaction mixture. The exchange half-reaction is used for studying in uitro the mechanism of action of the enzvme and has been shown to occur in hornogenacea and tissue slicer (331.
df
~
~
21 Metoholic incorporntion of trotope otomc without net sgnthe-
sis. By this mechanism the molecule containing isotopic atoms participates to a true and complete metabolic reaction and is
converted into other cellular compounds. However, during the metabolic transformation, the initial contribution of atoms of the labelled molecule to the final product is lost, even if its isotopic atoms remain in the molecule (32, 34). We have seen an example of this in the gluconeogenic pathway where the COz is incorporated for the oxaloacetate synthesis and then lost for PEP synthesis. However, because of the diearbonylie acid shuttle the CO? carbon lost is not always the same that was incorporated, and for this reason 14C-carbonatoms are found in glucose when ['"COz] is administered.
I I
Gluconeogenesis: A Teaching Pathway Criteria to establish a metabolic pathway
Gluconeogenesis is the metabolic pathway that synthesizes elucose from ovruvate. This uathwav is hioloaicallv .- . very important because in this way non-carbohydrate comvounds can he converted to elucose (1-4). Gluconeo~enesis has heen studied extensiveliin mammalian liver and it has been shown that the gluconeogenic glucose produced in this organ is essential for the life of the whole organism. In conditions of prolonged fasting, low carbohydrate diet or diabetes, nerves and blood cells which utilize primarily glucose as a source of energy, can not survive without a continuous supply of glucose produced by gluconeogenesis in the liver and kidney (3). The reverse of the glycolytic process-gluconeogenesiswas clearly recognized by Cori and Cori as early as 1929 ((21, see reference therein) but more than 30 years were necessary to reach a clear understanding of the whole pathway. During these years several schemes for the gluconeogenetic pathway were proposed and replaced with different ones as the experimental work proceeded, until the last scheme, which incorporates all the experimental results in a uniaue wav. As the studv of eluconeoaenesis proceeded, criterL for Goper experikentaf and for a correct evaluation of the data were developed. These criteria are of genera! meaning, and can he usefully applied to evaluate the validitv of other vathwavs and to direct the research for new pathways.- In this paper, the different schemes of gluconeogenesis, proposed at subsequent times during the elucidation of this pathway in mammalian and avian liver, will he reviewed. This may help in understanding how the criteria to be discussed have heen developed, and on which experimental data these criteria are hased. The first scheme to he proposed for a gluconeogenetic pathway suggested that the synthesis of glucose from pyruvate occurs by direct reversion of glycolysis (1, 2). Since all the reactions of glycolysis are shown to he reversible in uitro, the possibility of glucose synthesis in uiuo by this reaction was considered ((1.2) see also reference therein). Suhsequently, several experimental data indicated that alternative reactions catalyzed by enzymes not involved in glycolysis could participate in the synthesis of glucose from non-eluconeoeenic comuounds. The discoverv in liver cells of a specific gucose-6-dhosphate phosphatase and a specific fructose-1,6-diphosphate phosphatase suggests that these two reactions may participate in glucose synthesis from fructose-1,6-diphosphate( ( I ) , see reference therein). This proposed pathway has as intermediates the same exose vhosuhates of the alycolytic pathway, but utilizes two . . different enzymes. ~ u r & r eqe;imentsproved that the two specific phosphatases are key enzymes of gluconeogenesis, and that in uiuo the corresponding glycolytic reaction can not participate in the synthesis of glucose because it is thermodynamically feasible only in the direction of glucose degradation (1). Another reaction that earlier was thought to he irreversible in uiuo is the phosphorylation of pyruvate (1,5). In order to overcome this prohlem, alternative pathways for the synthesis of P E P (phosphoenolpyruvate) from pyruvate were proposed. The existence of these alternative
-
I am extremely grateful to Professor C. Baglioni for having read and corrected the manuscript. The work was supported by grant No. CT 75.00525.04. of Italian C.N.R. 86 / Journal of Chemical Education
GLYCOGEN
t t
GLUCOSE
t t t
PHOSPHOENOLPYRUVATE + CO,
PHOSPHOENOL-OXALOACETATE
FUMARATE
-
MALATE
11
ACETYLPHOSPHATE + CO.
LACTATE _ 2 PYRUVATE ~
+co,
OXALOACETATE
Figure 1. Scheme of carbohydrate synthesis from lactate and pyruvate according to Lipmann, 1941 (8)and (double line arrows)according to Solomon el al., 1941 (9).in the Lipmann scheme two acatylphosphate are necessary to produce one fumarate.Phosphoenol-oxaloacetate = CH-COOH
II
C-COOH
pathways is supported by some experiments that can not he explained by a direct phosphorylation of pyruvate to P E P via the pyruvate kinase reaction. I t has been shown that kidney cortex extracts catalyze the conversion of fumarate and malate to P E P (6) and that less than 2% of lactate carhoxyl carhon is converted to liver glycogen (7, 8). These data suggested that only carhon 2 and 3 of the lactate molecule participate to P E P synthesis from pyruvate and that dicarboxylic acids are metaholic intermediates. In 1941 Lipmann (9) proposed that the synthesis of gluconeogenetic P E P is initiated hy decarhoxylation and phosphorylation of pyruvic acid with formation of the two carbon unit acetyl phosphate; two acetyl phosphate molecules condensate to form fumarate; the dicarhoxylic acid is phosphorylated to give phosphoenol-oxaloacetate and then the phosphodicarboxylic acid is decarboxylated to form phosphoenolpyruvate (PEP) (Fig. 1). A short time later, Solomon et al. (9) by feeding rats with lactate and [14C] bicarbonate, established that the isotopic carhon is incorporated into glycogen; these authors suggested that COz is a gluconeogenic metabolite and proposed a pathway for the synthesis of P E P from pyruvate with a new reaction for the synthesis of the dicarhoxylic acid. Oxaloacetate is synthesized by direct carhoxylation of
pyruvate, converted to malate and then to fumarate; PEP is synthesized from fumarate through the reactions proposed by Lipmann (Fig. 1).Solomon et al: (9) point out that, because of the symmetry of the fumarate molecule, half of the labelled CO? incorporated into pyruvate is lost in the decarboxylation of fumarate and the other half is incorporated into glycogen. Topper and Hastings (10) using [14C] COz, [2-14C] pyruvate and [I-14C]acetate and looking at the position of the isotopic carhons incorporated into glucose units of glycogen (synthesized in vitro by rahbit liver slices) were ahle to establish that about 80% of the pyruvate molecules are randomized before being incorporated into these glucose units. These authors indicated that the carboxylation of pyruvate to form oxaloacetate and equilibration of oxaloacetate with symmetrical dicarboxylic acids (dicarboxylic acid shuttle) occurs four times as fast as the direct phosphorylation of the pyruvate. Topper and Hastings (10) point out that their isotopic experiments can not establish "whether phosphorylation of dicarhoxylic acid can or does occur." They believe that it is not necessary to postulate the formation of phosphoenolmalate orland phosphoenoloxaloacetate as intermediates for the PEP synthesis, because Lardy and Zigler (11)had shown that it is experimentally possible to synthesize PEP by direct phosphorylation of pvruvate using ATP and. as catalvst. kinase. . . . .Dvruvate -. The experiments of ' ~ o ~ and ~ e Hastings r (10) plus Lardy and Zigler (11) can be explained assuming that 80% of the pyruv& molecules enter in the dicarh~xylicacid shuttle before being phosphorylated and that the remaining 20% are incorporated into glucose without any change or loss of carbon atoms via direct phosphorylation (Fig. 2). In this scheme of gluconeogenesis, dicarhoxylic acids and COz do not have a role in the synthesis of PEP even if isotopic carbons of COz could be incorporated in the glucose unit of glycogen because of the shuttle. GLYCOGEN
t t
to eive PEP and COa. . (13). . The new scheme is orooosed on the- basis of known enzyme activities and of &e&bolic intermediates known to be normal constituents of the cell. However, this proposed pathway is thermodynamically unfavorable in the direction of phosphoenolpyruvate synthesis, not less unfavorahle than the direct reversion of the pyruvate kinase reaction (I). Utter and Kurahashi (11)and Krehs (I) indicate that a system of enzymatic reactions capable of maintaining a high NADPH/NADPC ratio in the cell would make energetically possible the PEP synthesis via the proposed pathway. Proofs of this possibility were given by Ochoa and coworkers (12). The proposed pathway bas no fumarate among its intermediates, hut a rapid equilibrium between malate and fumarate can be postulated. However, even if the new pathway seemed to give a logical explanation to all the experiments it was found incorrect after a few years. Mendicino and Utter (2, 15) found that isolated chicken liver mitochondria that lack nvruvate kiuase as well as malic enzyme, are able to synigesize PEP from pyruvate. This sueeests that in these mitochondria a oathwav for the PEP sy%hesis is present different from thk one proposed. In fact Utter and Keech (6) were ahle to detect in the chicken liver mitochondria a new COz-fixing biotine enzyme (16): the pyruvate carboxylase that catalyzes the carboxylation of pyruvate to oxaloacetate using ATP as energy donor. The discovery of the mitochondrial pyruvate carboxylase solves the problem of PEP synthesis in gluconeogenesis; Utter and Keech (18) proposed that pyruvate is directly converted into oxaloacetate by the pyruvate carboxylase reaction and oxaloacetate into PEP hy the already known phosphoenolpyruvate carboxykinase (Fig. 4). This pathway is the simplest mechanism for PEP synthesis among the ones previously proposed. It requires the breakage of two high energy bonds, and it makes the PEP synthesis from pyruvate thermodynamically feasible. In chicken liver, pyruvate carhoxylase and PEP COz kinase are both mitochondrial and the other enzymes necessary for the gluconeogen-
GLUCOSE
t t t t
-
PHOSPHOENOLPYRUVATE
..
PYRUVATE
+co,
OXALOACETATE
MALATE FUMARATE Figure 2. Scheme of carbohydrate synlbsis from pyrwate acwrding to Topper and Hastings, 1949 (10).
As suggested by Topper and Hastinge (lo), the problem of PEP synthesis from pyruvate can not he solved hy the isotopic techniques above indicated. In fact, only investigations directed at identifying the enzymes involved in the first step of gluconeogenesis will resolve this problem. In 1954 Utter and Kurahashi (11) and Krehs ( I ) proposed a new scheme for the PEP synthesis from pyruvate (Fig. 3). Pyruvate is converted to malate, malate to oxaloacetate, and oxaloacetate to PEP. This new scheme is in agreement with the known isotopic experiments and is supported by the discovery of the two enzymatic activities: malic enzyme, that catalyzes the carboxylation of pyruvate to malate using NADP as coenzyme (12). and phosphoenolpyruvate carboxykinase, that catalyzes the decarboxylation and phosphorylation of oxaloacetate by ITP (or GTP)
Figwe 3. Scheme 6f glucose synhsis hom lactate and pyrvvate according to M e r and Kurahashi. 1954 (11)and accoding to Krebs. 1954 ( 1 ) .
Volume 53.Number 2, February 1976 / 87
Figure 4. Scheme of glucose syntbis from pyrwate. The phosphoenolpyrwate synthesis from pyruvate is acmding to Uner and Keech, 1963 (17). The other Dans were from Krebs (3).
esis are localized in the cytoplasm. Therefore, during the gluconeogenesis P E P is synthesized inside the mitochondrion, diffuses in the cytoplasm, and in that compartment is converted to glucose (19). This pathway has been found valid for those liver cells in which pyruvate carhoxylase and P E P Con kinase are both localized in mitochondria. However, in liver of animals like rat, mouse, and hamster, pyruvate carboxylase is mitochondrial and P E P C02 kinase cytoplasmatic (20). Lardy and his school (21-23) have shown that a more complex pathway for P E P synthesis from pyruvate is then present (Fie. 5). In the livers of these animals, oxaloacetate synthe&d in the mitochondria can not diffuse in the cytoplasm because the mitochondria1 membrane is impermeable to this compound. Inside the mitochondria, oialoacetate is converted to malate or aspartate and these compounds diffuse into the cytoplasm where they are converted again to oxaloacetate. The conversion of oxaloacetate to malate and the diffusion of this dicarhoxylic acid into the cytoplasm has the double function of transporting outside the mitochondria oxaloacetate and "reducing power" (NADH) that can be utilized a t the level of the phosphoglyceraldehyde reaction. Aspartate is carrier of the sole oxaloacetate for the gluconeogenic pathway and it is utilized when the NADH for the eluconeoeenesis is svnthesized in the cvtoplasm. Other experiments prove that malic enzyme is not a gluconeogenetic enzyme but is primarily involved in liponeogenesis. Malic enzyme activity decreases in the liver of fasting rats, and increases when the animal is refed with a high carbohydrate diet (24). Furthermore, this enzyme has a high value of K , for NaHC03 (2). These data indicate that the malic enzyme works in the direction of pyruvate, Con, NADPH synthesis, and they also confirm indirectly that pyruvate carhoxylase is the COz fixing enzyme responsihle for dicarboxylic acid synthesis for gluconeogenesis. This history of the experiments performed t o elucidate the simple steps of gluconeogenesis is a good example of how advances in our understanding of biochemical pathways are
-
88 / Journal of Chemical Education
Figure 5. Scheme of glucose synthesis according lo Lardy, 1965 (21).
Figure 6.
E = Enzyme: A. B. C. D, and X = Metatolio intermediates.
obtained. Furthermore, these experiments point out the loeic of the metaholic pathway scheme and show also the techniques and criteria necessary to demonstrate a metabolic pathway at the molecular levcl. Criteria and Experimental Procedure to Establish a Metabolic Pathway
The structure of a metaholic pathway is very simple; it consists of a group of enzymes working in sequence in the sense that the product of the first enzvmatic reaction is substrate of thesecond and so on (Fig. 6): Each metaholic intermediate mav be a reaeent for several different chemical reactions. ~ o b e v e rit, Gill participate a t a verv. high - rate onlv in the reaction that is catalvzed hv a specific enzyme; even a weak covalent bond is rarely hroken by thermal motion within the cell, and reactions concerned with formation or breakage of covalent bonds are catalyzed bv enzyme (36). Each specific catalyst, increasing the ratd of the relative reaction, will contribute to the synthesis of the last product. Substrate specificity combined with the catalyzing power of the enzyme will make possible the rapid transformation of compound A into E with a rate consistent with the cell needs. In order t o prove the existence of a metaholic pathway it is necessary to demonstrate that all the enzymatic activities catalyzing the sequence of reactions and all the metaholic intermediates are nresent in a cell. These are necessary but not sufficient ckditions in order to demonstrate a metaholic nathwav. ". hut additional experimental proofs are required. I present here what I consider significant experimental proofs that must accompany the necessary conditions
+
(presence in the cell of the enzymes metabolites) in order to demonstrate a metabolic pathway. None of these proofs alone is sufficient to establish the existence of a metabolic pathway; however, these proofs reveal if a pathway is present and if i t can work in a cell. Free Energy Change of the Metabolic Pathway
The free energy change of the metabolic pathway provides the first indication that a pathway is thermodynamically feasible. I t can be calculated adding the free energy change of each reaction in the pathway (1,2,11,25-27). A preliminary indication of the energetic behavior of a pathway is obtained using free energy change in standard conditions or for a unique concentration value of all reagents and products of each enzymatic reaction in the pathway, and using pH, temperature, 02, and COz pressure values close to the physiological ones (1,2,11,25-27). This tvoe of calculation has been used bv Krebs (1.3) to postulat;?'that the exokinase, phosphof&ctokina&, A d ovruvate kinase reactions are irreversible. However. the z a n g e s of free energy calculated with unphysiologicalconcentrations of metabolites mav eive incorrect answers. A " typical example is the lactate dehydrogenase reaction (lactate NAD+ e Dvruvate NADH + H f ) that has a AGO = -15.54 kcal a n i a AG' = -6 kcal in the sense of pyruvate formation (AG' has been calculated with reagents and products in the concentrations of 0.01 mM a t 25°C and p H = 7). On the basis of this AG' value, the reaction in the cell should go only towards pyruvate NADH + H+ synthesis. However, i t has been proved by metabolic studies that in the liver cells this reaction can proceed in the opposite direction (3). I have calculated the AG of lactic dehydrogenase by the formula
-
+
+
+
AG
=
AGO + RTlog. [Pyruvate] [NADH] [H+] [Lactate] [NADf]
R is the gas constant, T is the absolute temperature, AGO value was taken from Burton and Krebs (25), the pyruvate and lactate concentrations and NAD+/NADH ratio were those determined in the rat liver cell (28, 29), and the H+ M. concentration was In this condition AG of the reaction in the direction of lactate formation is verv close t o zero (de~endineon the reagent and product concentrations found in the cells, AG varies between +0.34 and -0.95 kcallmole). Similar data have been obtained for the free energy change of the lactic dehydrogenase erythrocytes (38). These data indicate that the lactate dehydrogenase reaction is very close to thermodynamic equilibrium; hence, small changes in the concentration of the reagents andlor products c d a h i f t the reaction from one direction to the opposite. During active gluconeogenesis from lactate in liver cells, the reagents and products have t o be maintained a t certain steady concentrations that make thermodynamically possible the synthesis of pyruvate from lactate. Free enerev change calculations of each sten of ervthrocyte glycoly& (using physiological concentr&ions of the reagents and products) show that the maioritv of the metiholL reactions have negative AG (14). he ;her reactions have a very low, positive AG. The positive values of AG can be due to the technical difficulty of determining the actual content of the metabolite in the cell. I have repeated these calculations using the concentration of the glycolytic intermediates in the liver cells, and I found very similar results. These data show that three glycolytic reactions namely exokinase, phosphofructokinase, and pyruvate kinase have strong negative AG;all other reactions have AG very close to zero. The strone neeative value of the free-enerw variation of the erythrocse & hepatocyte glycolysis is prymarily due to the AG values of these reactions; there are reactions unique
to the glycolysis and irreversible in the cell. The other reactions in common with the glycolysis and gluconeogenesis have AG values verv close to zero and thev can oroceed in both directions. The AG (calculated for the actual concentrations of reagents and products) of each reaction in the pathway has t o be negative; if one reaction has nositive AG. even if the total ZG of the pathway is negativi, it indicates that the reaction is not in the pathway. The validity of the AG calculation is limited by the difficulty of determining the actual concentrations of reagents and products in the site where the reaction takes place in the cell. For thermodynamic reasons, in the cell the synthetic and the catabolic pathways of the same compounds are necessarily different. They may have some common reactions, but they necessarily differ for those reactions that have strong negative values of free-energy change. This is true for the svnthesis and deeradation of small molecular weieht " compounds (glucose, fatty acid, etc.) as well as for macromolecules (glycogen, proteins, RNA, DNA). Propertiesof the Enzyme
The enzymes that belong to a metabolic pathway show properties that are in harmony with the physiology of the pathway. Therefore, these properties can be used to verify whether or not an enzyme belongs to a pathway. Enzyme Specific Activity
The catalytic activity per g of fresh tissue (or per organ) of each enzyme in the metabolic pathway has to be equal to or higher than the metaholic rate of the whole pathway determined in uiuo as the rate of synthesis of the last metabolite (4). If an enzyme shows a specific catalytic activity lower than the rate of the pathway, this indicates that the enzyme can not be in the pathway. However, this criterion has to be used keeping in mind that i t is very difficult to estimate the real in uiuo catalytic activity of an enzyme. Furthermore. when the enzvme assav " is nerformed in crude preparations, other enzymatic reactions may interfere with it. On the other hand.. . purification causes losses of the enzyme (2,4). &
Variations of the Enzyme Catalytic Activity
When the metabolic rate of a whole pathway can be changed by altering the metabolic or hormonal conditions of the organism, the enzymes of the pathway should change their activity accordingly. When the catalytic activity of an enzvme decreases while the metabolic flow of the whole pathway increases, or vice versa, there is an indication that the enzvme is not in the nathwav (4.24). when the enzyme activity remains constant in value and the metabolic flow of the whole oathwav increases or decreases, no indication is obtained as to whether an enzyme belongs t o the pathway. The enzyme activity may remain constant because i t is high enough to support the increase in metabolic rate of the pathway, or i t does not decrease in the in uitro assay because the p&miological inhibitor or activator that operates in uiuo is lost or diluted during the isolation of the enzyme (2). Kinetic Properties of the Enzyme
The K , values for the substrates of an enzvmatic reaction assayed in both directions of a reaction, g&e useful indications for establishing which are the ~hvsioloeicalsubstrates of the reaction and from that, which>s thedirection of the reaction in the cell (2). The physiological substrates of an enzymatic reaction should have K , values related to their concentrations in the cell from which the enzyme is extracted. When the K , value of the suhstrate of an enzymatic reaction is much higher than the molarity of the substrate in the cell, this strongly suggests that it cannot be the substrate but is the product of the reaction. By this approach i t is possible to Volume 53, Number 2, February 1976 / 89
establish in which direction an enzymatic reaction proceeds in the cell. However. this criterion has to be used carefullv because kinetic experiments are made in conditions very different from the physiological ones (see previous paragraph); furthermore, i t is very difficult t o determine the actual concentrations of metaholites in the cellular compartment in which the enzyme is localized, particularly in the case of a metabolite not uniformlv distributed in the cell. The definition of the direction-of a reaction can be a key problem for the definition of a metabolic pathway, as has been seen for malic enzyme in the gluconeogenic pathway where no definitive information was obtained on the basis of free-energy changes (2). Other kinetic data may help to establish whether an enzvme belones to a metabolic ~ a t h w a vThe . effect of cellular metaholites as activators or inhibitors of the enzyme should be in harmony with the physiology of the pathway in which the enzyme is enclosed; e.g., inhibitory effects of Dhos~hataseand its AMP on the fructose-1.6-di~hos~hate activatory effect on the f r ~ ~ t o s e : b ~ h o ~ ~ kinase h a t eare in agreement with the concept that a high AMP level-associated with a low ATP level-stimulates glycolysis and inhibits gluconeogenesis and that fructose-1.6-di~hosphate phosphatase i s a gluconeogenetic enzyme and fructose-6phosphate kinase a glycolytic one (3,41. Specific inhibitors of the Enzyme
The use of specific inhibitors is a good test t o verify whether or not the enzvme belonrrs to a ~ a t h w a v The . administration in uiuo of compound, proved to be a specific inhibitor of an enzyme, must block the pathway a t the level of the reaction catalyzed by the inhibited enzyme (30, 31). As a consequence of this, the substrate(s) of the enzyme as well as its metabolic precursors will increase in concentration, whereas the enzyme product(s) and its metabolic derivatives will decrease in concentration. By this technique, called cross over-type plot, i t is clearly determined whether the inhihited enzyme belongs to a pathway; furthermore, the concentration changes of the cellular metabolites may provide information on other enzymatic reactions involved in the pathway.
a
Subcellular Locallzatlon of the Enzyme
When all the enzymes of the metabolic pathway are localized in the same cellular compartment, the product(s) of an enzymatic reaction will reach the enzyme that catalyzes the next reaction by free diffusion in the cell cytoplasm. When some of the enzymes of a metabolic pathway are localized in a subcellular compartment limited by a membrane and the remainine enzvmes are localized outside this membrane in another subcellular compartment, it is necessary to demonstrate that the metaholite that is synthesized in the first compartment and utilized in the second one can diffuse across the interposed membrane. If the membrane is impermeable to this metabolite the pathway is incorrect or incomplete. We have seen that in the rat the oxaloacetate which cannot diffuse through the mitochondrial membrane is converted to other metabolites which diffuse outside the mitochondria. These metabolites are converted again into oxaloacetate in the cytoplasm (21-23). In the rat the gluconeogenic pathway has two extra reactions with respect to the gluconeogenesis of other animals that have pyruvate carboxylase and phosphoenolpyruvate carboxykinase both localized in mitochondria.
- -
Use of Synthetic Substrates
Synthetic substrates can be added to biological systems (whole animals. ~ e r f u s e doreans. cell suspensions. tissue homogenates) in order to verify ifsnd how they are "tilized by the pathway that is under study. This approach is very powerful in the study of metabolism when metabolites containing isotopic atoms are used. The chemical properties of all the isotopes of an element 90 /
Journal of Chemical Education
are essentially the same, and molecules containing different isotopic atoms are metaholized bv the cell in the same way. Thiuse of isotopes thus allows study of the metaholic fate of compounds as well as the rate of their utilization in the cell. his experimental approach is particularly useful when a metabolic pathway is completely unknown. By this technique many p;oblemi of met&olisk have been solved and new pathways have heen discovered. We have discussed how with the use of isotopic carbon-carbonic anhydride i t has been established that COz fixation occurs in the eluconeoeenic nathwav (9). ~ k use e o f a compound: &iformly labelled in its carbon atoms oermits one to detect all the metabolic intermediates derivaied from the carbons of its skeleton. A more precise study can be made using the compound specifically labelled in single carbons. In this way it is possible t o follow the metaholic route of each carbon of the molecule and find out if the metabolic pathway is linear, branched, or a cycle. Using the pulse-chase technique i t is possible to determine which is the sequence of metabolites derived from the labelled precursors, in the order in which they are synthesized in the cell (35).In this technique a labelled precursor is administered t o a biological system and all the labelled metaholites present in the biological system are extracted and identified. The extraction of the labelled metabolites is carried out soon after the administration of the precursors and is repeated after short time intervals, sometimes after administration cf unlabelled orecursors (chase). The total isotopic content of the labelled metaboli& decreases with time, while the metaholites synthesized from it become labelled. The product of the first reaction that utilizes the labelled Drecursors, is the first t o become labelled and the first toieach a maximum of isotopic content; the other metabolites show similar behavior and by the time they become labelled and reach the maximal isotopic content, it is possible to determine the order in which they are synthesized. If the last metabolite is a final stable product, it increases constantly in isotopic content until all the radioactivitv of the nrecursor is contained in it. isotopes for metabolic studies may give erro~ k use e neous information when the transfer of isotooic atoms from one molecule to another occurs without a net conversion (32). This phenomena can take place by two different mechanisms 1) Erchanee reaction. It results from the oarticination of the malpcule containing isotopic atoms to a revemihle chemical system. In the cell this rystem can he an enzymatic reaetmn at equilibrium or an enzymatic.half reaction; in the first case the molecule containing isotopic atoms (substrate) is converted to product by a complete reaction and, because the reaction is at equilibrium, an equal amount of product present in the cell is reconverted to substrate. This oscillation permits the formation of labelled products without any net synthesis. In the second case, the molecule containing isotopic atoms participates to an enzymatic half-reaction. In this reaction an enzyme-substrate complex is formed but because of the absence of the other substrate(~)necessary for the complete reaction, the complex is hydrolyzed. However, the labelled product bound to the enzyme can exchange with a cold one added to the reaction mixture. The exchange half-reaction is used for studying in uitro the mechanism of action of the enzvme and has been shown to occur in hornogenacea and tissue slicer (331.
df
~
~
21 Metoholic incorporntion of trotope otomc without net sgnthe-
sis. By this mechanism the molecule containing isotopic atoms participates to a true and complete metabolic reaction and is
converted into other cellular compounds. However, during the metabolic transformation, the initial contribution of atoms of the labelled molecule to the final product is lost, even if its isotopic atoms remain in the molecule (32, 34). We have seen an example of this in the gluconeogenic pathway where the COz is incorporated for the oxaloacetate synthesis and then lost for PEP synthesis. However, because of the diearbonylie acid shuttle the CO? carbon lost is not always the same that was incorporated, and for this reason 14C-carbonatoms are found in glucose when ['"COz] is administered.
