Paper : 04 Metabolism of carbohydrates Module : 30 Principal Investigator, Paper Coordinator and Content Writer Dr. Ramesh Kothari, Professor UGC-CAS Department of Biosciences Saurashtra University, Rajkot-5 Gujarat-INDIA Dr. Vijaya Khader Dr. MC Varadaraj Content Reviewer: Prof. S. P. Singh, Professor UGC-CAS Department of Biosciences Saurashtra University, Rajkot-5 Gujarat-INDIA 1
Description of Module Subject Name Paper Name Module Name/Title 04 2
REGULATION OF CITRIC ACID CYCLE (KREBS CYCLEor TCA CYCLE) Overview of TCA cycle Aerobic organism uses a chain of chemical reactions known as citric acid cycleresulting in to generation of energy by the oxidation of acetate obtained fromproteins, carbohydrates and fats. Which is ultimately converted into CO 2 and energy in the form of ATP. It also provides precursors of certain metabolic substances i.e. amino acids with othernadh like reducing agent which are used in various other reactions. Citric acid cycle plays an important roleinvarious biochemical reactions. It indicates that TCAcycle was one of the earliest metabolic reaction and may have spontaneouslyoriginated in past. A type of tricarboxylic acid i.e. citric acid is used in this metabolic pathway hencethis cycle is known as citric acid cycle or tricarboxylic acid cycle or the Krebs cycle. Additionally acetyl-coa and H 2 O is consumed and NAD + reduces to NADH which produces CO 2 as a by-product waste.the NADH produced duringkrebs cycle is used into the ETC. The overall result of these closely linked metabolic reactions is the oxidized nutrients for the production of ATP. In eukaryotic cells,mitochondrion matrix is the place where the citric acid cycle occurs. Whereas in prokaryotic cells, like bacteria which lack mitochondria, these reactions takes place in the cytosol. 3
Overview of citric acid cycle regulation Regulation of TCA cycle is determined mainly by two things 1. Product inhibition and 2. Substrate availability. Overproduction of NADH and ATP like reduced coenzyme may result in to a waste of huge amount of metabolic energy, if the cycle allowed to run without any check point. ADP is other major substrate for the cycle. Which is then converted into ATP and a resulting reduced amount of ADP leads into accumulation of NADH, aprecursor moleculeby which a number of enzymes get inhibit. In the TCA cycle NADH isone of the product of all dehydrogenases with one exception that is succinate dehydrogenase. NADH inhibits several enzymes of TCA cycle viz. citrate synthase, α-ketoglutarate dehydrogenase, isocitrate dehydrogenase andpyruvate dehydrogenase. Pyruvate dehydrogenasealso gets inhibited by Acetyl-coAand α-ketoglutarate dehydrogenase and citrate synthasegets inhibited bysuccinyl-coa. Under in-vitro conditioncitrate synthase and α-ketoglutarate dehydrogenase gets inhibited byatp. Citrateact as feedback inhibitor for phosphofructokinase, an enzyme which is involved in glycolysis and catalyses the formation of a precursor of pyruvate i.e.fructose 1,6- bisphosphate. It prevents a constant high rate of flow when there is an accumulation of citrate with a decrease in substrate. 4
Calcium is also act as a regulator. Calcium level of mitochondrial matrix can increase during cellular activation which leads to the activation of pyruvate dehydrogenase phosphatase andalso activates the pyruvate dehydrogenase complex. Calcium also activates isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. Which results into increase in the rate of reactionof many steps in the cycle and ultimately increases flow throughout the pathway. *Additional Information: According to recent research, it has been demonstrated that there is an important connection between citric acid cycle intermediates and the regulation of hypoxiainducible factors (HIF). HIF has an important role in the regulation of oxygen homeostasis. It is closely associated with transcription factor whichglucose utilization, vascular remodelling, targets angiogenesis, apoptosis and iron transport. HIF synthesized constitutively and hydroxylation of even one of two critical amino acidprolineleads to their interaction with the von HippelLindau E3 ubiquitin ligase complex, leading them to rapid degradation. Prolyl 4-hydroxylases catalyses this reaction. Fumarate and succinate act as potent inhibitors of prolyl hydroxylases leading to the stabilisation of HIF. 5
Regulation of the TCA Cycle It is evident that allosteric regulators and covalent modification regulates severalimportant enzymes in metabolic pathways, whichensures the production of intermediate metabolites and all the final products in therequiredamount to maintain the cell in a steady stable state. Ultimately the wasteful production of intermediate metaboliteis avoided. C atom flows through TCA cycle under two level tight regulation: - first pyruvate converts into acetyl-coa, which isactually a starting material for the cycle and - second is the entry of acetyl-coa via reaction catalysed by citrate synthase Pyruvate is only one of the source for acetyl-coa, intermediates from other differentavailablepathways also plays an important role in the regulation of oxidation of pyruvate andtca cycle. α-ketoglutarate dehydrogenase andiso-citrate dehydrogenase reactions also regulates the citric acid cycle. 6
Regulation of Acetyl-CoA Production by the Pyruvate Dehydrogenase Complex In vertebrates regulation of the pyruvate dehydrogenase complex is carried out by both covalent and allosteric modification. ATP, acetyl-coa and NADH strongly inhibits the complex (Fig. 1). When long-chain fatty acids are available allosteric inhibition of pyruvate oxidation greatly increases. Too little input of acetate into the citric acid cycle leads to accumulation of AMP, CoA, and NAD +, all of which allosterically activate the pyruvate dehydrogenase complex. When enough amount of fatty acids and acetyl CoA is available this enzyme activity turns off, even in situation where ATP concentration and NADH/NAD + ratio is high enzyme activity turns off. This enzyme activity turns on under the condition where energy requirement is high and large flux of acetyl CoA is required. 7
Figure 1 Regulation of flow of metabolite from pyruvateviatca cycle. 8
In vertebrates pyruvate dehydrogenase complex, the allosteric regulation mechanisms are complemented bycovalent protein modification. As discussed earlier in addition to the enzymes, the pyruvate dehydrogenase complex also contains two regulatory proteins, these proteins have only purpose of regulation of the activity of the complex. Enzyme 1 gets phosphorylates and inactivated by a specific protein kinase, and the phosphate group is removed by hydrolysis by a specific phosphoprotein phosphatase, thereby activating Enzyme 1.ATP allosterically activateskinase: high level of ATP leads to the inactivation of pyruvate dehydrogenase complex by phosphorylation of Enzyme 1. Decrease in ATP level to the decreasing kinase activity and phosphatase action removes the phosphates from enzyme 1, thus activating the complex. In case of plants the pyruvate dehydrogenase complex is present in the mitochondrial matrix. Pyruvate dehydrogenase within chloroplast is inhibited strongly by NADH, act as a regulator. It is evident that reversible phosphorylation leads to the inactivation of enzymes found inplant mitochondrial matrix. InE. colipyruvate dehydrogenase complex is allosterically regulated same as that enzymes ofvertebrate.incase of bacterial enzyme regulation by phosphorylation do not occur. 9
Regulation of Three Factors of the TCA Cycle Metaboliteflow via citric acid cycle is regulatedandstrict, but not complex. Flow rate through the cycleis maintained by three factors: 1. Availability of substrate, 2. By accumulated products (Feedback Inhibition), and 3. Intermediates from later phase of cycle, allosterically inhibit early enzymes of the cycle. Three reaction catalyzed by citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase arestrong exergonic steps in the cycle and each of them can act as a ratelimiting step under specific situations. Acetyl-CoA and oxaloacetate are substrates for citrate synthase. Availability of these substrates varies with different metabolic conditions and sometimes also affects to the rate of formation of citrate. On the oxidation of isocitrate and α-ketoglutarate,nadh produces.this NADH accumulates under some conditions and when the ration of concentration of NADH to concentration of NAD + becomes large, both dehydrogenase reactions are highly inhibited. ATP act as an inhibitor of citrate synthase, while ADP act as an allosteric activator of citrate synthase. In vertebrate Calcium ions are the signal for contraction and it isassociated with increased demand for adenosine triphosphate, ultimately activatesisocitrate dehydrogenase and α-ketoglutarate dehydrogenase, with another complex of the pyruvate dehydrogenase. 10
Product of the first step of the citric acid cycle iscitrate. These Citrateact as an important allosteric inhibitor in the glycolytic pathway for phosphofructokinase-1. The Glyoxylate Cycle Phosphoenolpyruvateconverts into pyruvate and then conversion into acetyl-coa isirreversible.if acetate is converted to phosphoenolpyruvate. For the gluconeogenic pathway phosphoenolpyruvatecannot act as the raw material that leads to glucose. In absence of this mechanism, a cell or organism is not able to convert energy materials that degraded to acetate into carbohydrates. As per anplerotic reactionsthe reversible reaction that catalyzed by PEP carboxykinase,phosphoenolpyruvate can be synthesized. Oxaloacetate + guanosine triphosphatephosphoenolpyruvate + Carbon dioxide + guanosine diphosphate Because Carbon from acetate which enter into the TCA cycle appears after 8 steps later in oxaloacetate. Study of the stoichiometry of the cycle indicates that there is no conversion of acetate into oxaloacetate; for every cycle 2carbon enters as an acetyl-coa, which leave cycle as2co 2. Acetate serves both as an energy richmaterial and also as a source of phosphoenolpyruvatein plants and in few invertebrates and in some microbes. The glyoxylate cycle allows the net formation of oxaloacetate fromacetate is present in this organism. Few enzymes of the citric acid cycle works in 2different modein these organisms 11
1. Enzyme required for the oxidation of acetyl-coa to CO 2 can function in the citric acid cycle, as it occurs in most tissues, 2. The same enzyme can work as part of a specialized modification as the glyoxylate cycle. The glyoxylate cycle may have evolved before the TCA cycle and may have given rise to the TCA cycle. The overall reaction equation of the glyoxylate cycle, can be regarded as an pathway that forms the intermediate of TCA cycle, is 2 Acetyl-CoA + NAD + + 2H 2 O Succinate + 2CoA + NADH + H + 12
Glyoxylate Cycleis the Variation of TCA Cycle Oxaloacetate reacts with acetyl-coa and form citrate in the glyoxylate cycle exactly same as in the citric acid cycle.malate is formed when the glyoxylate condenses with acetyl-coa by a reaction catalysedwith malate synthase. Oxaloacetate is subsequently formed by oxidation of the malate, another round of the cycle starts when oxaloacetate condense with another molecule of acetyl-coa (Fig. 2). In each round of the glyoxylate cycle, net synthesis of one molecule of succinate occurs from two molecules of acetyl-co.remainingavailable succinate is used in different biosynthetic purposes. 13
14
Figure2The glyoxylate cycle and its relationship to the citric acid cycle. 15
Figure 3The reactions of the glyoxylate cycle in glyoxysomes withthe citric acid cycle in mitochondria, sharing intermediates through the cytoplasm. 16
Co-ordinate regulation ofglyoxylateand Citric Acid Cycles Glyoxylate and Citric Acid Cycles sharessome common intermediates whichnecessitates that these pathways are regulated and coordinated together. One of the crucial intermediate is Isocitrate, found at branching point amongstcitric acid and glyoxylatecycles (Fig. 4). Covalent type of modification of a protein kinase phosphorylates regulates the activity of Isocitrate dehydrogenase and ultimately the dehydrogenase getsinactivated. Inactivatedisocitrate dehydrogenase leads to the diversion ofisocitrateinto the glyoxylate cycle and fromthere it is diverted towards the glucose biosynthesis. The phosphate group is removed by phosphoprotein phosphatase from isocitrate dehydrogenase, which reactivates the enzyme with sending more isocitrate through the TCA cycle. Lowered concentration of regulators ofthese cycles, results into signalling enough flux through the TCA cycle andisocitrate dehydrogenase is inactivated by the protein kinase. 17
Figure 4Determination of the partitioning of isocitrate between the citric acid cycle and the glyoxylatecycle by regulation of isocitrate dehydrogenase activity. 18
Table 1List of allosteric effectors of kinase and phosphatase Intermediates of the Glycolytic pathway Intermediates of the Citric acid cycle Energy depletion indicating cofactors Phosphoenolpyruvate * Citrate AMP * Pyruvate * Isocitrate * ADP * 3-Phosphoglycerate * α-ketoglutarate * NADP + Fructose-6-phosphate Oxaloacetate * The kinase activity inhibited by all compounds shown in above table. Name having * stimulates the phosphatase activity. The intermediates that acts as an activator of isocitrate dehydrogenase also act as an allosteric inhibitors of isocitratelyase. Under the condition wherethe metabolisms yield is fast and provides sufficient energy to keep the concentrations of anaplerotic molecule low, leads to the inactivation ofisocitrate dehydrogenase. When isocitratelyase is gets free from inhibition and isocitrate enters into the glyoxylate pathway, there it is used for synthesis of amino acids,carbohydrates and other cellular materials. 19