Chapter 5 5.2 Metabolic pathways and metabolic control mechanisms 5.2.1 Revision of the reactions of the tricarboxylic acid(TCA)cycle a detailed revision of the TCA cycle is necessary to ensure an understanding of the mechanisms and reasons governing the choice of process conditions for encouraging production of any selected TCA cycle intermediate or related compound. Descriptions of the cycle can be found in many text books, for example in the open learning BIOTOL text entitled Principles of Cell Energetics. Chapter 7 of that text describe in great detail the TCa and glyoxylate cycles, both of which are relevant to this Chapter. The most relevant parts of the cycle are the control mechanisms and processes involved in the intermediary metabolism; these are influenced and exploite efforts to upset the balance of normal metabolism leading to overproduction of the desired organic acid Let us first examine Figure 5.1. It is worth spending some time on this in order to understand the rationale of the remainder of this Chapter glycolysis Glycolysis(the Embden Meyerhof pathway)is a ten enzyme pathway which is summarised in Figure 5.1. During the course of this pathway, glucose is cleaved to two pyruvate molecules-a process involving the utilisation of two aTP(generating two ADP)but later there is formation of four ATP from four ADP. thus a net yield of two ATP is achieved. Another consequence of glycolysis is the reduction of two molecules of nicotinamide adenine dinucleotide (NAD)to NADH H. Although the exact mechanism of the individual reactions of glycolysis is not really necessary for this Chapter, we must bear in mind the fact that during the process energy and reducing power are formed. The two reactions indicated by *are two further enzymatic steps which, along with three reactions of the TCA cycle, constitute the glyoxylate cycle In the reaction that bridges glycolysis and the TCA cycle, for each pyruvate degraded to acetyl CoA, one Coz is released and a further nad is reduced to nadh+ A variety of starting materials other than glucose or its derivatives is possible for use by some micro-organisms; the four shown in Figure 5.1 are all initially converted to acetyl CoA for entry into the central metabolic pathways
120 Chapter 5 5.2 Metabolic pathways and metabolic control mechanisms 5.2.1 Revision of the reactions of the tricarboxylic acid (TCA) cycle A detailed revision of the TCA cycle is necessary to ensure an understanding of the mechanisms and reasons governing the choice of process conditions for encouraging production of any selected TCA cycle intermediate or related compound. Descriptions of the cycle can be found in many text books, for example in the open learning BIOTOL text entitled 'Principles of Cell Energetics'. Chapter 7 of that text describe in great detail the TCA and glyoxylate cycles, both of which are relevant to this Chapter. The most relevant parts of the cycle are the control mechanisms and processes involved in the intermediary metabolism; these are influenced and exploited in efforts to upset the balance of normal metabolism leading to overproduction of the desired organic acid. Let us first examine Figure 5.1. It is worth spending some time on this in order to understand the rationale of the remainder of this Chapter. Glycolysis (the Embden Meyerhof pathway) is a ten enzyme pathway which is summarised in Figure 5.1. During the course of this pathway, glucose is cleaved to two pyruvate molecules - a process involving the utilisation of two ATP (generating two ADP) but later there is formation of four ATP from four ADP. Thus a net yield of two ATP is achieved. Another consequence of glycolysis is the reduction of two molecules of nicotinamide adenine dinucleotide (NAD') to NADH i H'. Although the exact mechanism of the individual reactions of glycolysis is not really necessary for this Chapter, we must bear in mind the fact that during the process energy and reducing power are formed. The two reactions indicated by * are two further enzymatic steps which, along with three reactions of the TCA cycle, constitute the glyoxylate cycle. In the reaction that bridges glycolysis and the TCA cycle, for each pyruvate degraded to acetyl &A, one COZ is released and a further NAD+ is reduced to NADH + H+. gtycobsis A variety of starting materials other than glucose or its derivatives is possible for use by some micro-organisms; the four shown in Figure 5.1 are all initially converted to acetyl CoA for entry into the central metabolic pathways
The large scale production of organic acids by micro-organisms glucose 2 ADP 2 NAD glycolysis 2 ATP 2 NADH +2H NADH +H eacton fatty acid malate Isocitrate the TCA fumarate a-oxoolutarate Figure 5. 1 A simplified diagram of glycolysis and the CA)cycle showing the entry points for various substrates. 'indicates the two to the glyoxylate cycle Compounds in boxes are p otential substrates for entry 5.2.2 The relationship between anabolism and catabolism At this point we need to consider the two halves of metabolism -anabolism and catabolism- and in particular the metabolic control involved catabolism Catabolism is the process by which intra-or extracellular molecules are degraded to rield smaller ones which are either waste products or building blocks for biosynthesis During catabolism reducing power in the form of NADH+ H, FADH or NADPH +H is generated. Subsequent reoxidation of these cofactors(particularly NADH) by aerobic cells releases energy which is converted to ATP, the major short term energy storag currency. The mechanisms involved in this aTP formation are the electron transport chain and oxidative phosphorylation. These processes are intimately linked-a bit like two parts of a zip fastener-in that when oxidation of NADH takes place, the formation of 3 ATP from 3 ADP usually takes place
The large scale production of organic acids by micro-organisms 121 ~~ Figure 5.1 A simplified diagram of glycolysis and the triirboxylic acid (TCA) cycle showing the entry points for various substrates. indicates the two reactions specific to the glyoxylate cycle. Compounds in boxes are potential substrates for entry into the TCA cycle, via acetyl CoA. 5.2.2 The relationship between anabolism and catabolism At this point we need to consider the two halves of metabolism -anabolism and catabolism - and in particular the metabolic control involved. Catabolism is the process by which intra- or extracellular molecules are degraded to yield smaller ones which are either waste products or building blocks for biosynthesis. Jhmg catabolism reducing power in the form of NADH + H', FADH2 or NADPH + H' is generated. Subsequent reoxidation of these cofactors (particularly NADH) by aerobic cells releases energy which is converted to ATP, the mapr short term energy storage cumncy. The mechanisms involved in this ATP formation are the electron transport chain and oxidative phosphorylation. These processes are intimately linked - a bit like two parts of a zip fastener - in that when oxidation of NADH takes place, the formation of 3 ATP from 3 ADP usually takes place. catabolism
anabolism Anabolism is the building up or biosynthesis, of complex molecules such as protein, nucleic acids and polysaccharides, from raw materials originating from intra-or extracellular sources. The biosyntheses are energy (aTP)requiring processes. Catabolism and anabolism have to be carefully regulated and are inevitably intimately t are the three areas where the processes of catabolism and anabolism are Catabolism produces ATP, reducing power and intermediates. Anabolism requires all three, thus these are the three main links. No living cells can store large amounts of ATP. There is a finite amount of adenine distributed between AMP, ADP and ATP. Thus if the cell has a relatively high concentration of atP, the concentrations of AMP and/or ADP must be lowered. The balance alters like a"see-saw", as one goes up the other must come down. In addition the total amount of nad/NADH and NadP/NAdPH in the cell is constant. What is the advantage of the see-saw type of change to the ratio of the The answer is that such a system is far more sensitive to small changes in concentration of the respective compounds. The cell is recognising a change of the ratio of compounds, rather than the rise or fall of a single compound Although cells cannot store ATP, they must always have a minimum amount available to keep them alive. Thus a constant level of ATP must be maintained indicating that catabolism and anabolism occur constantly under normal conditions ∏ One compound controls the overall metabolism of the cell regulating and balancing anabolism and catabolism. Can you name it? The answer in practice is aTP though you would have been theoretically correct if you had said ADP and AMP. Indirectly, NAd or NADH are also compounds which regulate the anabolic/catabolic balance The metabolic control is exercised on certain key regulatory enzymes of enzymes called allosteric enzymes. These are enzymes whose catalytic activity is hrough non-covalent binding of a specific metabolite at a site on the protein the catalytic site. Such enzymes may be allosterically inhibited by aTP or all activated by ATP (some by ADP and /or AMP). Thus atp is the effective controller of metabolism but because AMP ADP + AtP is constant, it is really the ratio of adenine nucleotides which is important. This ratio is energy charge termed the adenylate charge or energy charge and is expressed as Energy charge 0.5 [ADP]+ [ATPI [AMP]+[ADPI+ [ATPI
122 Chapter 5 anabolism albsteric enzymes Anabolism is the building up or biosynthesis, of complex molecules such as protein, nucleic acids and polysaccharides, from raw materials originating from intra- or extracellular sources. The biosyntheses are energy (ATP) requiring processes. Catabolism and anabolism have to be carefully regulated and are inevitably intimately linked. What are the three areas where the processes of catabolism and anabolism am n linked? Catabolism produces ATP, reducing power and intermediates. Anabolism requires all three, thus these are the three main links. No living cells can store large amounts of ATP. There is a hite amount of 'adenine' distributed between AMP, ADP and ATP. Thus if the cell has a relatively high concentration of ATP, the concentrations of AMP and/or ADP must be lowered. The balance alters like a "see-saw", as one goes up the other must come down. In addition the total amount of NAD+/NADH and NADP+/NADPH in the cell is constant. What is the advantage of the see-saw type of change to the ratio of the n concentrations? The answer is that such a system is far more sensitive to small changes in concentration of the respective compounds. The cell is recognising a change of the ratio of compounds, rather than the rise or fall of a single compound. Although cells cannot store ATP, they must always have a minimum amount available to keep them alive. Thus a constant level of ATP must be maintained indicating that catabolism and anabolism occur constantly under normal conditions. One compound controls the overall metabolism of the cell regulating and n balancing anabolism and catabolism. Can you name it? The answer in practice is ATP though you would have been theoretically corred if you had said ADP and AMP. Indirectly, NAD+ or NADH are also compounds which regulate the anabolic/catabolic balance. The metabolic control is exercised on certain key regulatory enzymes of a pathway called allosteric enzymes. These are enzymes whose catalytic activity is modulated through noncovalent binding of a specific metabolite at a site on the protein other than the catalytic site. Such enzymes may be allosterically inhibited by ATP or allosterically activated by ATP (some by ADP and/or AMP). Thus ATP is the effective controller of metabolism but because AMP + ADP + ATP is constant, it is really the ratio of adenine nucleotides which is important. This ratio is termed the adenylate charge or energy charge and is expressed as: 0.5 [ADP] + [ATP] [AMP] + [ADP] + [ATPI Energy charge =
The large scale production of organic acids by micro-organisms The theoretical limits are 1.0 (all ATP)and 0(all AMP)with a normal working range of 0.75 to 0.9. The involvement of energy charge in the integration and regulation of metabolism is considered further in the BIOTOL text entitled 'Biosynthesis and the Integration of Cell Metabolism After revising the tCa cy ctions in more detail we shall return to the subject of metabolic control by ATP Figure 5.2 shows a detailed version of the TCA cycle indicating cofactor changes and the individual intermediates ace h) A NADH+H malate cIs-aconrtate fumarate f FAD NADH +H succinate CoASH a-oxoglutarate GTP. NAD ADP succinyl CoA NADH +H ATP CoA+3 NAD++ FAD+ADP + Pi 2 CO +3 NADH +3H++FADH +ATP +CoASH Figure 5.2 The tricarboxylic acid cycle Enzymes: a)citrate synthase; b )aconitase; c)isocitrate dehydrogenase; d)a-oXoglutarate dehydrogenase; e) succinyl CoA synthetase; f) succinate dehydrogenase; g)fumarase; h) malate dehydrogenase; i) nucleoside diphosphokinase 5.2.3 The control of metabolism In section 5. 2. 2 we considered a simple equation expressing the energy charge of the cell in terms of the ratio of adenine nucleotides. Figure 5.3 summarises the principal allosteric enzymes of glycolysis and the tCa cycle and indicates how the individual adenine nucleotides influence the activity of a variety of enzymes. The enzymes to the right of the glucose to pyruvate pathway are those involved in glycolysis; those to the eft are involved in gluconeogenesis, ie the synthesis of glucose from pyruvat
The large scale production of organic acids by micro-organisms 123 The theoretical limits are 1.0 (all ATP) and 0 (all AMP) with a nod working range of 0.75 to 0.9. The involvement of energy charge in the integration and regulation of metabolism is considered further in the BIOTOL text entitled 'Biosynthesis and the Integration of Cell Metabolism'. After revising the TCA cycle reactions in more detail we shall return to the subject of metabolic control by ATP. Figure 5.2 shows a detailed version of the TCA cycle indicating cofactor changes and the individual intermediates. Figure 5.2 The tricarboxylic acid cycle. Enzymes: a) citrate synthase; b) aconitase; c) ismitrate dehydrogenase; d) a-oxoglutarate dehydrogenase; e) succinyl CoA synthetase; f) succinate dehydrogenase; g) fumarase; h) malate dehydrogenase; i) nucleoside diphosphokinase. 5.2.3 The control of metabolism In section 5.2.2 we considered a simple equation expressing the energy charge of the cell in terms of the ratio of adenine nucleotides. Figure 5.3 summarises the principal allosteric enzymes of glycolysis and the TCA cycle and indicates how the individual adenine nucleotides influence the activity of a variety of enzymes. The enzymes to the right of the glucose to pyruvate pathway are those involved in glycolysis; those to the left are involved in gluconeogenesis, ie the synthesis of glucose from pyruvate-
124 Chapter 5 glucose ATP Inhibition by I glucose-6-phosphate AD glucose-6-phosphate Stimulated by AMP ATP and ADP ADP fructose-1, 6-biophosphate Stimulated by glucose- ATP by ATP and NADH pyruvate NAD+ buta a strand NADH +H+ NADH haloacetate V nhibited by ATP Isocitrate lated by AMP ADP inhibited by NADH a-oXoglutarate Stimulated by ADP nhibited by NADH Figure 5. 3 Major control points of glycolysis and the TCA cycle. Enzymes: I, hexokinase: Il, phosphofructokinase; Ill, pyruvate kinase; IV, Pyruvate dehydrogenase; V, citrate synthase; VI aconitase; VI, isocitrate dehydrogenase; Vl, a-oXoglutarate dehydrogenas
1 24 Chapter 5 Figure 5.3 Major control points of glycolysis and the TCA cycle. Enzymes: I, hexokinase; 11, phosphofructokinase; 111, pyruvate kinase; IV, pyruvate dehydrogenase; V, citrate synthase; VI, aconitase; VII, isocitrate dehydrogenase; VIII, u-oxoglutarate dehydrogenase