Monosaccharides, and particularly glucose, owe their importance to the fact that their oxidation provides the living organisms with a major part of the energy they require. Furthermore, the carbon atoms of glucose can be found in a large number of compounds (amino acids, fatty acids, sterols, glycerol, etc.).
In the study of the metabolism of carbohydrates we will therefore come across substances belonging to proteins, lipids or nucleic acids; this shows that any division of cellular metabolism would be arbitrary, because the latter forms a set of well integrated reactions and it should therefore be borne in mind that divisions are made only for facilitating the presentation.
Contents
Digestion and Absorption of Carbohydrates:
Only monosaccharides pass easily through the cell membranes of living organisms. But the carbohydrates present in the food of animals or microorganisms are often disaccharides like lactose and sucrose, and polysaccharides like starch and glycogen. They will have to be hydrolyzed before they are absorbed and metabolized.
I. Digestion of Carbohydrates:
The hydrolysis of polysaccharides is catalyzed by exoenzymes, i.e. enzymes liberated outside the cells in which they were synthesized. We will study here only the hydrolysis of the polymers of glucose — starch and glycogen — as it takes place in the intestine of man; the other polysaccharides are generally hydrolyzed only by microorganisms (example: hydrolysis of cellulose by bacterial cellulases).
Starch and glycogen are hydrolyzed by amylases, enzymes catalyzing the scission of α-1, 4-glucosidic linkages; two of them are known:
1. β-amylase, is mainly found in plants, especially in starch-storing grains; it attacks starch from the non-reducing end of chains by detaching maltose units, which, although in the a-form in the polysaccharide, are liberated in the form of β-maltose: α-Glc-(1→ 4)-β-Glc;
2. α-amylase is formed during the germination of cereals. Besides, it is present in saliva and pancreatic juice. It also breaks the α-1, 4 glucosidic linkages, but at any place inside the polysaccharide molecule; in the case of linear chains (amylose), total hydrolysis into maltose units is finally obtained.
But in the case of amylopectin and glycogen, the hydrolysis of α-1, 4-glucosidic linkages is not complete near the branch points and besides, theα-1, 6 glucosidic linkages which constitute these branch points are not attacked. A mixture of α-maltose: α-Glc-(1→ 4)-α-Glc and residual dextrins composed of branched oligo- and polysaccharides is obtained.
Amylo-α-1, 6 glucosidase, secreted by the cells of the intestinal mucosa, and also called “debranching enzyme”, enables the hydrolysis of these residual dextrins by catalyzing the hydrolysis of the α-1, 6-glucosidic linkage of the branch points.
Lastly, the hydrolysis of polysaccharides is completed by a α-glucosidase, present in the membrane of the brush-shaped border of enterocytes, called maltase, which splits the maltose units (resulting from the action of α-amylase and oligo-α-1, 6 glucosidase) giving 2 molecules of glucose.
Furthermore, two other enzymes are found in the intestine; they are also membranous, and attack the disaccharides:
i. Invertase (β-fructosidase) catalyzing the hydrolysis of sucrose into glucose + fructose.
ii. Lactase (β-galactosidase) catalyzing the hydrolysis of lactose into galactose + glucose.
Finally, the digestion of carbohydrates included in diet mainly leads to glucose (from starch, glycogen, lactose, sucrose), galactose (from lactose) and fructose (from sucrose); the problem to be examined is therefore the absorption of these monosaccharides.
II. Absorption of Monosaccharides:
The absorption of monosaccharides through the intestinal mucosa does not take place by simple diffusion. Various hexoses which diffuse at comparable rates are absorbed at different rates; some pentoses are absorbed more slowly than glucose or fructose although their smaller molecular weight enables them to diffuse more rapidly; lastly, the absorption of monosaccharides proceeds from a compartment where concentration is low, towards a compartment of higher concentration.
These observations can also be generalized, because they apply to the absorption of substances other than monosaccharides, and to absorption through cell membranes in general.
Membranes have complex structures which confer on them a selective permeability, they allow certain substances to pass through them and oppose the passage of other compounds. The concentration of a product on one of the sides of the membrane is a process which requires energy; for this reason it is spoken of as active transport.
The mechanisms of cellular absorption of carbohydrates is not yet fully understood. It is however known that glucose and galactose cross the membrane of the intestinal epithelial cell with the help of a Na+ dependent carrier.
On the other hand, in some cases, especially among microorganisms, it was possible to show the participation of an enzymatic type of protein called permease, specific of a sugar and this is possibly a general phenomenon.
In any case, specificity is an important characteristic of absorption. This is confirmed by observations made with insulin, a hormone secreted by the pancreas and which — through a phenomenon which is not sufficiently known — promotes the absorption of glucose; as a matter of fact, a lack of insulin affects the absorption of glucose but not that of fructose.
Once absorbed, monosaccharides will proceed towards the liver through the portal vein.
Metabolism of Monosaccharides:
In the following discussion we will mainly refer to glucose.
As mentioned below, the other monosaccharides (especially fructose and galactose) are readily converted into derivatives of glucose and their metabolism links up with that of glucose:
I. Phosphorylation of Glucose:
As just mentioned, the phosphorylation of monosaccharides, the first step of their metabolism, takes place very rapidly after their absorption, and we will see that the metabolism of monosaccharides mainly calls upon the phosphorylated monosaccharides.
The phosphorylation of glucose in the cell cannot take place by direct reaction between the monosaccharide and the phosphate ion because the equilibrium is too unfavourable. A phosphate group is given by ATP; there is breakdown of a phosphoanhydride linkage and formation of a phosphoester linkage (see fig. 4-20).
The equilibrium of this reaction is largely in favour of the formation of the ester; it is practically irreversible, because it is highly exergonic (ΔG0 # – 5 kcal/mole). This is an example where the energy of ATP is utilized for the formation of a compound which would be very difficult to synthesize otherwise; we will see many other reactions of this kind.
This reaction consists of a transfer of the phosphate group, it is a transphosphorylation, catalyzed by a transphosphorylase or kinase, here a glucokinase, catalyzing specifically the phosphorylation on the alcoholic hydroxyl carried by the carbon 6 of glucose.
Some organisms possess less specific enzymes; for example, yeast has a hexokinase which catalyzes the phosphorylation of glucose, fructose and mannose into the corresponding hexose-6-phosphate.
Besides, some monosaccharides like galactose are phosphorylated on their hemiacetal group. There are therefore C1– and C6– hexokinases. It will be noted that Mg2+ is necessary for the reaction; this ion is very often the co-factor in the reactions involving phosphorylated compounds.
In most cases, the action of the ion consists in maintaining the ATP molecule in a compact conformation which is generally the physiologically active form of this compound.
Glucose-6-phosphate (or glucose-6-℗) thus obtained, occupies a central position in the metabolism of carbohydrates; starting from this compound, various metabolic pathways are possible; they are represented diagrammatically in figure 4-21.
Before taking up the study of these diverse metabolic pathways, one should try to answer the following question: why would the molecules of glucose-6-℗ take up one of these paths rather than another, in other words, what decides the fate of the glucose-6-℗ molecules?
This is a problem of regulation which we will consider again at a later stage, but it may be already stated that the proportion of molecules of glucose-6-℗ taking one of these metabolic pathways is variable in a given organism or even in a given cell, depending on the physiological state of this organism or cell.
Thus, when large quantities of ATP are necessary, a major proportion of molecules of glucose-6-℗ will undergo oxidation (generating ATP, as will be seen in the following); furthermore, there will be hydrolysis of the reserve polysaccharides and formation, from lipids, of larger quantities of compounds which may be oxidized.
On the contrary, when ATP requirements are diminished, a large proportion of molecules of glucose- 6-℗ will be converted into glycogen; besides, a major fraction of compounds formed intermediately during the first steps of oxidation of glucose-6-℗ will be directed, not towards the subsequent steps of oxidation (which are the most efficient for the production of ATP), but towards the synthesis of lipids.
II. Formation of Glucose from Glucose-6-℗:
Phosphorylated monosaccharides cannot pass through the cell membrane and enter the external medium; they must first lose their phosphate group. We know this cannot take place by the reverse reaction of phosphorylation.
The hydrolysis reaction of the ester linkage is catalyzed by an enzyme calledglucose-6-phosphatase, absent in many cells (which cannot therefore liberate glucose and have no reason for doing so), but present in hepatic cells which must liberate glucose into the blood for its distribution to other tissues.
Synthesis and Degradation of Polysaccharides:
I. Synthesis of Glycogen (Glycogenogenesis):
The synthesis of glycogen takes place gradually with addition, at each step, of a new glucose unit which attaches itself by its hemiacetal group to the alcoholic hydroxyl of another glucose unit situated at the end of a pre-existing glycogen chain. But the direct reaction between a hemiacetal group and an alcoholic OH does not take place spontaneously (the equilibrium is not favourable); for this reason; the hemiacetal group must first be activated, i.e. made more reactive in view of the formation of the glucosidic linkage.
This activation proceeds in 2 steps:
1) Transformation of glucose-6-℗ into glucose-1-℗, catalyzed by phosphoglucomutase;
2) Formation of uridine-diphosphate-glucose (UDPG) by action of uridine-triphosphate (UTP) on glucose-1-℗. The energy required for the synthesis of the glucosidic linkage is supplied by UTP, while in general, mostly ATP yields energy for the biosynthesis reactions. But ATP acts indirectly to reconstitute UTP from UDP formed in the reaction (see diagram; fig. 4-23).
Then UDPG transfers the glucose portion of its molecule to a pre-existing glycogen chain, lengthening the latter by one unit (reaction catalyzed by glycogen synthetase).
These 3 reactions are represented diagrammatically in figure 4-22.
It must be noted that glycogen synthetase catalyzes only the formation of 1, 4 glucosidic linkages. The branches are due to a branching enzyme capable of transferring an oligosaccharide fragment (4 glucose units for example), collected from a longer linear chain, to the carbon 6 of a glucose of this linear chain.
The synthesis of starch and cellulose in plants takes place by similar mechanisms, with activation of glucose in the form of ADP-glucose in the case of starch and GDP-glucose in the case of cellulose.
II. Degradation of Glycogen (Glycogenolysis):
It does not take place in the reverse direction of synthesis, but by phosphorolysis (a reaction where the phosphate ion plays a role similar to that of water in hydrolysis).
The reaction is:
The glucose units are detached by rupture of the α-1, 4-glucosidic linkage at the non-reducing end of the chain. Having examined the synthesis of glycogen (see fig. 4-22), we observe that the last glucose unit incorporated in the polymer is the first to be detached.
The reaction is reversible in vitro, and glucose units can be added to a pre-existing chain, but this mechanism is not the physiological process of glycogen synthesis.
Glucose-1-℗ formed in the reaction is transformed into glucose-6-℗ by a reaction which is the reverse of the one seen in the previous paragraph, by the action of phosphoglucomutase.
The glycogenolysis reaction is catalyzed by phosphorylase a, the active form of glycogen phosphorylase, the formation of which (from phosphorylase b, inactive or of low activity) is influenced by a hormonal control whose mechanisms is devoted to metabolism regulation (see fig. 8-15).
The pathways of the synthesis and degradation of glycogen can be summarized in a simple diagram (see fig. 4-23). Instead of referring to it merely as glycogen (which suggests a well-defined molecule) it would perhaps be preferable to talk of glycogen molecules because there are molecules of different dimensions; besides, depending on the physiological conditions, these molecules will sometimes lengthen by successive additions of glucose units (when the energetic requirements are low) and sometimes shorten by successive removals of glucose units (when the ATP requirements are high).
We have seen that glycogen molecules are synthesized and degraded by different processes. This is a rather common phenomenon which we will observe again especially in connection with the synthesis and degradation of fatty acids, and which presents the very great advantage of enabling a better regulation of cellular metabolism.
One of the most important possibilities of control lies in the regulation of the activity of enzymes, which can be either activated or inhibited. If the cell used the same enzyme (or enzymes) to catalyze the synthesis and catabolism of a substance, an activator (or inhibitor) would have an identical effect on the two opposite processes and the result would be nil.
On the contrary, if the pathways of synthesis and degradation utilize different enzymes, an activator or inhibitor could affect one of them without affecting the other.
For example, the degradation of glycogen can be more pronounced when there is an increase in the quantity of phosphorylase a, the active form of the enzyme, which happens (as mentioned above) by the effect of hormones like adrenaline and glucagon; this leads to an increased formation of glucose-6-℗; this action on phosphorylase affects only the glycogenolysis and has obviously no effect on the synthesis of glycogen because the phosphorylase has no part in it.
Glycolysis:
It is usually considered that oxidation of glucose consists of 2 series of steps: the first is extra-mitochondrial and leads to the production of 2 molecules of pyruvic acid; the second is intramitochondrial and permits the complete oxidation of pyruvic acid to CO2 and H2O.
The first series of reactions is called glycolysis, or Embden-Meyerhof glycolytic pathway, or anaerobic phase of the oxidation of glucose (because these reactions can take place in the absence of oxygen).
As will be seen in the following discussion, this series of reactions can also, in certain cases, lead to the formation of lactic acid or ethyl alcohol (the terms lactic fermentation or alcoholic fermentation are then used sometimes). We will now consider these glycolytic reactions and indicate the main characteristics of some of them.
Energy Balance of Glycolysis:
While drawing this balance, one should bear in mind that one molecule of fructose-1, 6-di-℗ gives 2 molecules of triose-℗ and that thanks to triosephosphate-isomerase, these 2 molecules can continue to follow the pathway of glycolysis.
1. In Aerobiosis:
The transformation of one molecule of glucose into 2 molecules of pyruvic acid is therefore accompanied by a gain of 8 molecules of ATP.
2. In Anaerobiosis:
We know that the electron carrier system does not function, that NADH is reoxidized during the formation of lactic acid or ethanol, and that consequently the corresponding energy is not utilized for the synthesis of ATP. Therefore, only 4 ATP are formed (and 2 ATP used), i.e. a gain of 2 A TP only per molecule of glucose.
It is very clear that the energy which can be stored by the cells (in the form of ATP) from one molecule of glucose in the course of the Embden-Meyerhof’s sequence is distinctly greater (4 times) when glycolysis takes place in aerobiosis. We will see in the following that, when oxygen is available, oxidation can continue (through Krebs cycle) and that it is then accompanied by a considerable gain of ATP.
Only some microorganisms can manage with anaerobic glycolysis to meet their energy requirements. In animals (and man) this process functions only for limited times, till oxygen is again available; the terminal products of anaerobic glycolysis — lactic acid and ethanol — are also comparatively toxic, and if their concentration exceeds a particular threshold value, they arrest the metabolism in the cells concerned.
At the very beginning of this century, Hopkins had observed that the muscle can contract (which requires energy) in anaerobiosis and that — in such conditions — lactic acid is produced in much larger quantities than if the muscle worked in presence of oxygen and is accumulated till the exhaustion of the muscle; he had further observed that if oxygen is supplied to this exhausted muscle, the lactic acid disappeared and the capacity of the muscle to contract was restored. We will see in the following how, in aerobiosis, lactic acid can be reconverted into glucose or glycogen.
Metabolism of other Monosaccharides in Relation with Glycolysis:
We will see briefly how other monosaccharides can join the glycolytic pathway and also how they can be formed from glucose by mechanisms called “conversion of monosaccharides”.
Some organisms, especially yeast, possess a hexokinase capable of phosphorylating glucose, fructose and man- nose to the corresponding hexose-6-℗. Fructose-6-℗ is an intermediate of glycolysis. As for mannose-6-℗, we have seen (in the study of phosphohexose isomerase, that it could, just as its epimer glucose-6-℗, be isomerized to fructose-6-℗.
Lastly, we will make a quick study of the principal reactions of the metabolism of galactose, because on the one hand, it represents a comparatively frequent source of carbohydrates in diet (it is produced by the hydrolysis of lactose or milk sugar), and on the other hand, it will provide an example of metabolic disorder.
We will recall that D-galactose is an epimer of D-glucose; this will enable us to study the transformations of this monosaccharide without the necessity of writing the structures of all the compounds.
1) Galactose is First Phosphorylated by ATP in Presence of a Kinase:
2) Then, UDP-glucose: α-D-galactose- 1-phosphate uridyl-transferase (also called phosphogaluctose-uridyl-transferase) catalyzes the following reaction:
galactose -1-℗ + UDP – glucose ←→ UDP – galactose + glucose-1-℗
As may be seen in figure 4-33, this reaction consists of an exchange between galactose-1-℗ and the glucose-1-℗ involved in UDP-glucose, the mode of formation of which was already described (see fig. 4-22).
This UDP-galactose is either incorporated in glycoproteins and various sphingoglycolipids like the cerebrosides (galactolipids) and the gangliosides or converted by a 4-epimerase into UDP-glucose by a simple change of configuration at carbon 4 (and it then follows the metabolism of glucose).
But in infants suffering from a genetic disease called galactosemia, UDP- glucose: α-D-galactose- 1-phosphate uridyl-transferase is absent and the new born is therefore incapable of metabolizing galactose-1-℗ because reaction 2 cannot take place (we have denoted this by a bar across the arrow).
Grave disorders appear (intestinal disorders, icterus, cataract, etc.) which can even lead to death if milk lactose is not immediately replaced by another sugar. This hereditary disease is due to an alteration of the gene of the transferase, which is therefore absent in these individuals.
The reversible conversion reaction of galactose into glucose with monosaccharides in conjugated form with a nucleotide (reaction no. 3 above) is a particular example of a very general mechanism. It is in the conjugated form that monosaccharides are transported, converted into one another, or incorporated into molecules of polysaccharides (see for example, the biosynthesis of glycogen, fig. 4-22).
The following are a few examples of these conjugated forms of monosaccharides:
UDP-Glc (or UDPG), UDP-Gal, UDP-Glc NAc, UDP-Glc AU, GDP- Man, GDP-Fuc, CMP-NeuAc, ADP-Glc (or ADPG).
Some reactions of conversion of conjugated monosaccharides are described in the following diagram:
Biosynthesis of Oligo and Polysaccharides:
I. From “Free” Carbohydrates:
The biosynthesis of polysaccharides rarely takes place from free carbohydrates.
The following examples may however be given:
1) Transfer of short chains (about 7 residues of glucose conjugated by α-1, 4 linkages) to growing glycogen molecules catalyzed by a branching enzyme which grafts by α-1,6 linkages.
2) Action of levan saccharose (or levan sucrase) which synthesizes levans (or fructosans) by the following mechanism:
Sucrose + (Fructose)n → (Fructose)n+1 + Glucose
or, dextran saccharase (or dextran sucrase) which leads to dextrans by a similar mechanism:
Sucrose + (GIucose)n → (Glucose)n+1 + Fructose
II. From Monosaccharide 1-Phosphates:
Phosphorylases can catalyze in vitro, the formation (or lengthening) of polysaccharide chains from glucose-1-℗ and thus enable the synthesis of glucosans in which the glucose residues are joined by α-1, 4 linkages. However this mechanism does not seem to occur in physiological conditions.
But the following synthesis of sucrose has been described, catalyzed by sucrose-phosphorylase
Glucose-1-℗ + Fructose ←→ Sucrose + Pi
III. From Nucleotides – Monosaccharides:
The pathway of the synthesis of oligo- and polysaccharides most commonly followed by the cells is the one which uses the nucleotides-monosaccharides, a partial list of which was given above. The transfer of the monosaccharide is performed by transferases which bear the name of the carbohydrate they conjugate: galactosyl-, mannosyl-, N-acetylglucosaminyl-, sialyl- (or neuraminyl-), glucuronosidyl-transferases, for example.
The glycosyl-trans- ferases involved in the biosynthesis of glycans of glycoproteins are enzymes integrated in the luminal face of membranes of the endoplasmic reticulum.
For information, we will restrict ourselves to the description of the biosynthesis of a disaccharide, the lactose (see fig. 4-46) on the one hand, and on the other hand, of the more complex process (see fig. 4-47) of the biosynthesis of the biantennate glycan of the N-acetyl-lactosaminic type.
1) Synthesis of Lactose:
The synthesis of lactose in the mammary gland is performed by the transfer of galactose from UDP-Gal to glucose, catalyzed by a galactosyl transferase and necessitating the presence of α-lactalbumin, the milk protein. The association of these two proteins constitutes the lactose synthetase system.
2) Synthesis of a Glycan of the N-Acetyl-Lactosaminic Type:
It was long believed that the residues of monosaccharides were conjugated one after the other by direct transfer from glycosylnucleotides as in the case of O-glycosylproteins. In fact, the biosynthesis of a glycan of the N-acetyllactosaminic type results from a complex “maturation” process passing through structures of the oligomannosidic type; the various phases of the process are illustrated in figure 4-47.
A lipidic intermediate A, glycosyl-dolichylpyrophos- phate, is first synthesized and then transferred completely by an oligosaccharide transferase to an acceptor protein, giving rise to glycoprotein B (see fig. 4-47). The glycan of the latter is then degraded by specific glycosidases acting in the order indicated by the circled numbers.
The elimination, by α-glucosidases, of residues of glucose leads to a glycan of oligomannosidic structure of the type existing in unit A of thyroglobulin. The action of α-1, 2 mannosidases located in the Golgi apparatus provides the glycoprotein C; the structure of the glycan thus formed was characterized in various glycoproteins such as ovalbumin. N- acetylglucosaminyl transferase I then forms glycan D.
Next, α-mannosidases lead to the compound E on which N-acetylglucosaminyl transferase II conjugates a second residue of N-acetylglucosamine (compound F). The N-acetyl- lactosaminic type glycan, which is bi-antennate in the example chosen, is finally completed thanks to the successive action of galactosyl- and sialyl-transferases.
The reactions leading to structures A and B take place in the granular reticulum; the N-glycosylation of a protein is therefore a co-translational phenomenon.
On the contrary, the reactions leading to structure C and then to the building of N-acetyl-lactosaminic type glycans take place in the smooth reticulum. They are therefore post-translational. In the case of O-glycosylproteins, the biosynthesis of glycans takes place entirely in the smooth reticulum and is consequently entirely post-translational.
As for the glycoproteins thus synthesized, either their peptide chain will be liberated from the ergatoplasmic membrane and they will be secreted out of the cell, or they will remain integrated in it and externalized by the membrane flow, becoming the constituents of the plasmic membrane.
Lastly, the glycans of oligomannosidic structure can be phosphorylated in the position 6 of some mannose residues and, according to a mechanism proposed by Sly, the residues of mannose-6-phosphate of glycans carried by the lysosomial hydrolases then represent the signal which permits the segregation of theses enzymes from the “externalized” glycoproteins of the cell.
As a matter of fact, only the glycoproteins which carry the mannose-6-phosphate signal will be firmly attached to a receptor of the Golgi membrane and their maturation towards a N-acetyl-lactosaminic structure will be blocked.
They will be still present on the internal face of membranes of vesicles which are then formed and which will become primary lysosomes. On the contrary, the non- phosphorylated glycoproteins evolve towards the N-acetyl-lactosaminic type by the cascade of transformations described in figure 4-47.
The set of mechanism of intracellular “distribution” of glycoproteins are diagrammatically represented in figure 4-48. They demonstrate the fundamental importance of glycan structures as recognition signals.
