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Charles Saladino Introduction This lecture will cover some of the main metabolic pathways associated with carbohydrate metabolism. There is a defined and important inter-relationship between the pathways with homeostatic balance being maintained through various types of regulation, including covalent, allosteric, hormonal, and second messenger systems. These pathways will include glycogenesis and glycogenolysis, gluconeogenersis, glucose isomer metabolism, and the pentosephosphate pathway, otherwise known as the hexosemonophosphate Shunt, and the Kreb’s cycle. Let us begin with the pentosephosphate pathway, often given too little time in introductory biochemistry courses. The structures of the pathway can be found on pages 449 & 650, Devlin) of your text. You do not have to memorize the pathway, but you do need to understand its significance and the material I will now cover. Hexosemonophosphate Shunt or Pentose Phosphate Pathway (PPP) Purpose of this pathway is two-fold. The first is the production of NADPH (a reducing agent and the reduced form of NAD+ used in many processes, such as fatty acid synthesis. This can be considered a cosubstrate for key enzymes. The second importance of the PPP is that it generates intermediate metabolites essential for the synthesis of nucleic acids and nucleotides. Because this pathway represents an alternative to glycolysis, be sure to reacquaint yourself with the oxidation of glucose to pyruvate. The PPP occurs in two phases, an oxidative phase and a non-oxidative phase. Again, I refer you to your text as we start with the oxidative phase. Here, glucose-6-P is the starting metabolite. Remember that this has been formed by the action of the phosphorylating enzyme, glucokinase. There are three steps during this first phase that result in the generation of the D isomer of ribulose-5-P. Of importance to note is the formation of NADPH + H+ from NADP+ in steps 1 and 3. Notice that in the non-oxidative phase, both isomerization and condensation reactions of several sugar molecules occur. I am not happy with the figure presentation in your text, so I will attempt to clarify it. First, however, let’s look for the most significant intermediates generated by this non-oxidative phase. These products are ribose-5-P (used for nucleic acids), fructose-5-P, and glyceraldehyde-3-P (G-3-P). (Remember G-3-P from glycolysis?) Now besides the G-3-P, ribose-5-P, and fructose-6-P, the non-oxidative phase generates the interconversion of 3, 4, 6, and 7-carbon sugars. Some of these enter glycolysis. We also see that the PPP pathway provides a means for cleaving the carbon chain, one carbon at a time. However, a big however, unlike the Kreb’s cycle, this pathway does not occur as a series of consecutive reactions leading from glucose-6-P to six CO2 molecules.
I now give you a diagram below, where the two, 5-carbon isomerization products generated from ribulose-5-P (xyulose-5-P and ribose5-P – not shown below, but shown in your text) interact with a transketolase enzyme to form two products. One is the 3-carbon G-3-P, the other is a 7-carbon sugar (sedoheptulose-7-P), shown below. The G-3-P formation is very important, because it can re-enter glycolysis.
Now, the G-3-P and the seduloheptose, catalyzed by a transaldolase, join and rearrange to
form two new sugar products, fructose-6-P (6 carbons) and erythrose-4-P (4 carbons). (By the way, you should be keeping track of carbons during these reactions.) The final piece of the puzzle is in the lower right part of the above diagram. Notice that the 4-carbon erythrose-4-P and the 5 carbon xyulose-6-P join and rearrange (catalyzed by a transketolase) to form fructose-6-P and G-3-P. Both are glycolytic intermediates. You should also note that transketolases will transfer 2-carbon units, whereas transaldolases transfer 3-carbon units. Each of these carbon units is transiently attached to the enzyme in the course of the reaction. Please also note that these reactions of the PPP pathway are all reversible. That means that the equilibrium can shift in the direction which provides the product when it is in demand. Just by way of one of many examples, take the situation where more ribose-5-P is needed than NADPH – as in a cell actively engaged in mitosis. Here is what will happen: Most of the glucose-6-P is converted into fructose-6-P and glyceraldehyde-3-P (G-3-P) by glycolysis. Then the transketolase and transaldolase convert two molecules of fructose-6-P and one molecule of G-3-P to yield three ribose-5-P molecules by reversal of the non-oxidative phase of the PPP pathway described above. You could summarize this by the following reaction (Note the 30-carbon balance on both sides of the equation.): 5 glucose-6-P + ATP -----> 6 ribose-5-P + ADP + H+ So what would be the predominant reaction if NADPH were required in great amounts, as would be the case in a demand for fatty acid, cholesterol, neurotransmitter, and nucleotide biosynthesis? The answer is the oxidative phase of the PPP pathway. Glucose-6-P + 2NADP+ + H2O <=====> ribose-6-P + 2NADPH + 2H+ + CO2 Coordination of Glycolysis and the Pentose Pathway Obviously by now you realize the glucose-6-P is metabolized by both glycolysis and the PPP pathway. So how can we partition this metabolite between the two pathways? The cytoplasmic concentration of NADP+ plays a key role in determining the fate of glucose6-P. The first reaction in the oxidative phase of the PPP pathway is essentially irreversible, and under physiologic conditions serves as a control site. The most important regulatory factor is the level of NADP+, because this is the electron acceptor in the oxidation of glucose-6-P. Low levels of this key cofactor are made worse by the fact that NADPH competes with NADPH+ in binding to the enzyme. The ratio of NADP+ to NADPH in the cytosol of the liver of a well-fed rat is 0.014. This low ratio ensures that NADPH generation is tightly coupled to its utilization in reductive biosynthesis (ex fatty acid synthesis). In other words, excess NADP+ will allow NADPH to go anywhere. This all translates into the fact that the non-oxidative phase of the PPP pathway is controlled by the availability of NADP+. Some Final Thoughts on the Pentose Pathway
Please take note that by the time the ribulose-5-P has been produced from glucose-6-P, we have gone from a hexose to a pentose by way of a decarboxylation at step three of the oxidative phase. Another aside: Now here is an opportunity for you to have done some thinking. You probably learned in your pre-requisite (I hope) that when mitochondria are involved, oxygen is required. The fact that oxidation occurs in the cytosol tells us that this process is not dependent upon the mitochondria or upon the Kreb’s cycle. Please remember that for next week. You might have heard of the Calvin Cycle in plants. Here, the cycle begins with CO2 and goes on to use NADPH in the synthesis of glucose. In the pentose pathway, we begin with the oxidation of a glucose-derived carbon to CO2 to yield NADPH. Further, the Calvin cycle converts 6-carbon and 3-carbon molecules to the starting material, ribulose-5-P. The pentose pathway converts 5-carbon sugars (ribose-5-P) into 6-carbon and 3-carbon intermediates of glycolysis. In photosynthetic organisms, the enzymes are the same as in mammalian organisms. Some would call this evolutionary economy. What an interesting stroke of creative genius. Glycogen Metabolism Glycogen can be considered a readily available storage form of glucose. This glycogen structure is a very large, branched glucose residue polymer that can be degraded to indirectly provide glucose when it is required for energy production. Although the majority of glucose residues are linked by α-1,4-glycosidic bonds, branches occur about every ten residues, due to α-1,6glycosidic bond formation. This bond – like that formed in joining two amino acids – is a dehydration synthesis reaction, where water is removed to form a bond. On your own, look up the structure of these two bond types, as well as a β-glycosidic linkage, and please know them for the next exam. They are easily found in the Devlin text or on line. I will note here that α-glycosidic linkages form open helical polymers, but β-glycosidic linkages produce almost straight chain polymers to form fibrils, as is seen in cellulose, for example. Can you suggest for the Discussion Board the advantage(s) of glycogen having branches, instead of being a long polymer? Biochemically, glycogen is not as reduced as are fatty acids. Then why would we store glycogen at all? We could just convert excess energy into fatty acids. Right? Well, there are several answers to that question. The first is that in between meals, the controlled breakdown of glycogen and subsequent release of a glucose product makes that sugar available to those organs where it is needed. So we might think of glycogen as a buffer against non-homeostatic plasma glucose levels. This is especially important for the brain, whose only source of energy is glucose under-non-starvation conditions. Further, mobilization of glucose stores (as glycogen) is quick and is a good source of
energy for sudden bursts of activity. Finally, glucose can supply at least some energy anaerobically, unlike fatty acids which require the oxygen found in mitochondria. The liver and the skeletal muscle are the two major storage sites of glycogen. However, there is a greater glycogen concentration in the liver (potentially 7-10%) vs. that of skeletal muscle (about 2%); but because of its much greater mass, more glycogen overall is stored in muscle than in the liver. Microscopically, glycogen is found in the cytosol as granules ranging in size from 10 – 40 nm. Another point of importance is that the glycogen stored in muscle is regulated to meet the energy needs of that itssue, where liver glycogen degradation and synthesis are balanced so as to maintain plasma glucose levels for the good of the whole organism. At this point we need to clarify something. Although I (and many texts) talk of making glucose available from glycogen, the breakdown of glycogen does not yield a glucose molecule directly. The degradation process is basically a three-step process: 1) glucose-1-P is released from glycogen; 2) glycogen as a substrate is “remodeled” to allow further degradation, as it is during synthesis; 3) glucose-1-P is further converted into glucose-6-P for further metabolism. This overall breakdown process is called glycogenolysis. Observe this on page 630-631 of your text. Then look at Figure 15.51 to observe glycogenesis. You are responsible for the names of the respective enzymes for the next exam. The glucose-6-P product of glycogenolysis basically has three fates: 1) it can continue into glycolysis; 2) it can enter the now-familiar-to-you pentose pathway to generate NADPH and ribose derivatives; 3) it can be converted to glucose to be released into the blood stream. Now in both the degradation and the synthesis of glycogen, we take note of two modified glucose molecules. These are UDP-glucose and UTP-glucose. The “U” stands for uridine, the DP is diphosphate, and the TP is triphosphate. This is the same U as is found in RNA nucleotides. Please note their respective roles in both process, and if you have any questions about this, let me know via email or the Discussion Board, as you are, at least in general, responsible for their respective roles, as it pertains to this discussion. You would, however, expect a phosphate to be available to help drive the energy-requiring synthesis of glycogen. As a point of interest, until not long ago, it was presumed that blood glucose was the sole precursor for glycogenesis. However, under physiological conditions, it is now known that most the new glycogen is formed from not from plasma glucose, but from gluconeogenic (glucose-producing) precursors, especially lactate and alanine. Both of these molecules are easily converted into glucose in the liver. Please read page 620-621 of your text to find out the relationship of this gluconeogenic lactate to the Cori cycle. There is also an alanine cycle which sends alanine from the muscle cells to the liver. The final topic for glycogen is its regulation. This is quite complex. Various enzymes that participate in glycogen metabolism allosterically respond to metabolites that signal
the cell’s energy needs. In other words, enzyme activity is adjusted to meets the needs of the cells in which the enzymes are present. Glycogen metabolism is also hormonally regulated, in order to allow glycogen metabolism to respond to the needs of the whole organism. My apologies, but you need to know the main features of Figure 15.5415.55 in order to appreciate some of this regulation. Then email me to explain how the insulin/glucagon ratio directly affects the events shown in the figure. I want some mechanism described here, please. Feeder Pathways into Glycolysis The purpose of this section is to account for the fate of two important monosaccharides after they have been digested and absorbed. These two sugars are galactose and fructose. The increase in fructose in the American diet (whatever that is) has been meteoric over the last couple of decades. This is because of a variety of reasons, not the least of which is that it is a very inexpensive commodity. In terms of metabolic health and efficiency, this huge increase in the use of fructose is doing none of us any good. Galactose Metabolism Please read your text on the Absorption, Transport, and Distribution of galactose and fructose. Note that they are not absorbed in the same manner, with galactose utilizing the same Na+-dependant carrier system as glucose does. Obviously, a major source of dietary galactose is lactose, especially from milk and milk products. Other sources include the cell turnover, especially cell membranes, upon which lysosomes work to provide glycoproteins and glycolipids from those membranes. Like fructose and glucose, galactose must be phosphorylated before it can be mobilized. This takes place in the liver. Galactose kinase is the enzyme responsible for this phosphorylation, and it costs one ATP. The product is galactose-1-P. Refer to the figure below to follow this pathway, and please pay close attention to the color scheme, so that you will more easily understand what is happening. First, the newly generated galactose-1-P can not enter glycolysis until it is first converted to UDP- galactose. This occurs in an exchange reaction. In this reaction, the enzyme galactose-1-P uridyl transferase transfers the removal of UMP (shown in black) from UDP-glucose and adds it to the galactose-1-P to form UDP-galactose (shown in red and black), leaving behind glucose-1-P (shown in blue). Notice two things that are happening. First, UDP galactose can under go an isomerization reaction, using the enzyme UDP-galactose 4-epimerase. This reaction regenerates UDP-glucose, which is recycled back to start a new set of reactions. Note the efficiency of this, as opposed to using a totally different pathway to generate all the UDP
glucose needed for this reaction sequence – which brings us to a the second point. The formation of glucose-1-P is now positioned to be converted into glucose -6-P (by another isomerization reaction using phosphoglucomutase) or even glucose by removing the phosphate group using the enzyme glucose-6-phosphatase. The glucose itself can be utilized in a variety of ways, including being exported to other tissues from the liver. On the other hand, the newly-generated glucose-6-P is a metabolite that is part of glycolysis, and hence it can be incorporated into that pathway for oxidation to pyruvate. There are certainly some side issues here. For example, if energy is abundant, say from a high caloric intake, the glucose-1-P might be channeled into glycogenesis instead of glycolysis. This type of common sense thinking is what I hope you will strive to achieve. However, the bottom line of what we have shown here is that (and how) the carbons of galactose can wind up in glucose for glycolysis, because there are no catabolic pathways to metabolize galactose.
Fructose In most organisms, like galactose and other hexoses (such as mannose), fructose can enter glycolysis after conversion to a phosphorylated derivative. D-fructose is found in many fruits, but is unfortunately loaded into our diets in a variety of ways, from syrups to
candies etc. Whereas galactose and glucose are absorbed into the intestinal mucosa by way of an active, energy-requiring transporter with a countercurrent uptake of sodium, fructose uptake utilizes a far less efficient, sodium-independent transporter (GLUT-5) for absorption. All three monosaccharides require another transporter (GLUT-2) to then enter the portal circulation. Here we must make a clear distinction between the metabolic events taking place in the liver vs. those in the muscle. In the liver, the fructose-1-P pathway is utilized. This is
illustrated in this Figure to the right. The first step involves the phosphorylation of fructose to fructose-1-P, using the enzyme fructokinase and at the cost of one ATP. This six-carbon product is then split into two, three-carbon sugars, dihydroxyacetone phosphate (DHAP) and glyceraldehyde by the enzyme B aldolase. Note that the DHAP is a glycolytic intermediate. A triose kinase then converts the glyceraldehydes to glyceraldehyde-3-P, also a glycolytic intermediate and at the cost of one ATP. By this means, fructose can enter glycolysis. In the liver, fructose can theoretically be converted to fructose-6-P by a glucokinase enzyme. However, the affinity of the glucokinase is 20 times greater for glucose than for fructose. Thus, very little fructose-6-P is formed in the liver, because glucose is so much more abundant in this organ. Only a heavy intake of fructose would activate this pathway. Besides, in the liver, glucose is the preferred fuel, as is the case in muscle. In the muscle, fructose can be converted directly to fructose-6-P at the cost of an ATP. This is already an intermediate of glycolysis, although again, glucose is the preferred fuel. There has been a ten-fold increase in fructose consumption in the last 25 years in the United States, due to the high use of high-fructose corn syrup, which is sweeter and is cheaper to produce than glucose. Although we will not discuss carbohydrate regulation until the next lecture, I will note here that fructose catabolism in the liver by passes the phosphofructokinase catalyzed step of glycolysis, wherein fructose-6-P is converted into fructose-1,6-bisphosphate. This is a highly regulated metabolic control point. This could potentially disrupt fuel metabolism, whereby glycolysis would be directed toward lipid
synthesis, if ATP production were not needed. Importantly, herein might be a link between fructose consumption and obesity that is ever-increasing in the United States.