1 BI25M1 METABOLIC REGULATION LECTURE 1 AIM: To review: how balance and economy are achieved by metabolic regulation; how some metabolic regulation control over [enzyme]. involves [Lehninger Edition 4 pp. 21-22; 225-233; Chapter 15 Lehninger Edition 5 pp. 20; 220-227; Chapter 15 Instant Notes Sections C5, J7] 2 Metabolism is the flow of energy (and matter) from the environment to the organism, through the organism, and from the organism back to the environment. The flow occurs through pathways of enzyme-catalysed reactions. [Energy Transformations Lecture 2] By and large, organisms remain fairly constant in composition, from whole-body to molecular levels, through long periods of their life. This stability (called ‘homeostasis’ by biologists and ‘dynamic steady state’ by physicists/chemists) doesn’t mean that nothing is happening. 3 Energy and matter are continually flowing (as noted above), and molecules and cells are continually lost and replaced (i.e. are ‘turned-over’). [Energy Transformations Lecture 2] So, although outwardly more-or-less unchanging, organisms, in fact, are seething with chemical reactions. How is such stability achieved? Answer: any change in one part of the metabolic flow system must 1 be detected; 2 trigger a counter-balancing flow change elsewhere in the flow system. 4 A simple example (at the whole-body level): in an organism exhibiting homeostasis, the rate of energy and matter flow INTO the organism is balanced by the rate of flow OUT OF the organism. If the rate of flow IN changes (e.g. increases), this is detected, and triggers a counterbalancing (i.e. increased) change in the flow OUT, 5 so that no net change occurs in the composition of the organism itself. So, metabolism is regulated to achieve balance. [In growing cells and organisms, the flow IN is greater than the flow OUT, and in ill and dying cells and organisms, the flow IN is less than the flow OUT.] As well as balance, metabolic regulation achieves economy. Particular numbers of cells and molecules are needed by an organism at any time. Production of more would be uneconomic, and is prevented by metabolic regulation. 6 An example (at the molecular level): A fairly constant [isoleucine] is needed in a protein-synthesising cell. At this concentration, isoleucine acts as an inhibitor of the first reaction in the metabolic pathway leading to its own synthesis. threonine isoleucine This ‘feed-back inhibition’ [BI1505 Enzymes lecture BI25M1 Enzymes Lecture 4] means that the required [isoleucine] is maintained in the cell. Notice how [isoleucine] is involved in both the detection system and the triggering system mentioned earlier. 7 So, as Lehninger (third edition, p.12) says: “Living cells are self-regulating chemical engines, continually adjusting for maximum economy.” 8 REGULATION OF METABOLISM OCCURS MAINLY THROUGH CHANGES IN ENZYMES Although [substrates], [products], [co-reactants] pH, temperature and other factors may affect rates of enzyme-catalysed reactions, [BI25M1 Enzymes lectures; BI25M1 Practical 1] it is through changes in the enzymes themselves that changes in rates of metabolic reactions are mainly brought about. There are two ways in which this occurs: 1 changes in concentrations of enzymes; 2 changes in activities of enzymes. 9 CHANGES IN METABOLIC REACTION RATES THROUGH CHANGES IN ENZYME CONCENTRATIONS The rate of an enzyme-catalysed reaction is proportional to [enzyme], at least, under particular conditions. Thus, in the presence of a fixed, high [substrate], an increase in [enzyme] should increase the reaction rate, while a decrease in [enzyme] should decrease it. So, in some circumstances, changes in flow rates through metabolic pathways might be brought about by changes in [enzymes]. 10 In fact, the two main ways in which changes in enzyme concentration are brought about are: 1 changes in expression of the gene encoding the enzyme; 2 zymogen activation. 11 CHANGES IN ENZYME CONCENTRATION BROUGHT ABOUT BY CHANGES IN GENE EXPRESSION Changes in [enzyme] occur just as do changes in the concentration of any biomolecule. Thus, an increase in the rate of synthesis or a decrease in the rate of degradation of a biomolecule leads to an increase in [the biomolecule], while a decrease in the rate of its synthesis or an increase in the rate of its degradation leads to a decrease in its concentration. In practise, changes in rates of synthesis are particularly influential. 12 Enzymes are proteins, and the main point at which control over the rate of synthesis of proteins is exerted is transcription. The switching on of the lac operon in E. coli [BI20M3 Transcription lectures] is an example of a change in the rate of flow through a metabolic pathway that is caused by a change of [enzyme], which itself is caused by a change in the rate of enzyme synthesis produced by a change in the rate of transcription of the gene encoding the enzyme. 13 increased [lactose] outside E. coli cell lactose enters cell allolactose formed repressor of lac operon deactivated by allolactose transcription of lac operon unlocked increased rate of synthesis of mRNA encoding -galactosidase (transcription) increased rate of -galactosidase synthesis (translation) increased [-galactosidase] increased rate of lactose catabolism 14 So, the increase in [lactose] is detected, and triggers an increase in flow through the lactose catabolism pathway. A look back at the process: a change in [-galactosidase] is caused by a change in the rate at which it is synthesised, which itself is produced by a change in the rate of transcription of the -galactosidase gene. This is an effective, but coarse form of regulation, because: 1 it takes time for it to begin to operate (mRNA needs to be made and then translated); 15 2 when increased flow is no longer needed, the system takes time to stop (mRNA and protein need to be degraded); 3 it is really an on/off switch rather than a fine-tuning control (in the absence of lactose, there is no flow; the presence of lactose switches on the flow). This type of regulation is, therefore, a way of responding to a fairly long-term change in the environment of the organism by producing a fairly long-term change in the pattern of its intracellular metabolism. 16 CHANGES IN ENZYME CONCENTRATION BROUGHT ABOUT BY ZYMOGEN ACTIVATION Zymogens are inactive precursors that, when cleaved, become active enzymes. [Examples include enzymes used in protein digestion How Organisms Handle Nitrogen Lecture 2] A particular example: dietary protein enters stomach stomach cells stimulated to secrete the hormone gastrin stomach cells stimulated to secrete pepsinogen pepsinogen cleaved to pepsin (autocatalytically, and, later, by pepsin itself) dietary protein cleaved by pepsin 17 So, the increase in [protein] is detected, and triggers an increase in flow through the protein catabolism pathway. A look back at the process: a change in [pepsin] is caused by a change in the rate at which it is made from its pre-formed, inactive precursor. This is an effective, but still rather coarse form of regulation: 1 it doesn’t require mRNA synthesis or translation (a store of zymogen is ready for activation when needed); 18 2 once activation begins, further activation is encouraged (as pepsin generated acts on remaining pepsinogen), so a ‘snowballing’ effect occurs; 3 however, the regulation is really an on/off switch (in the absence of dietary protein, there is no flow; the presence of protein switches on the flow). This type of regulation, like the last, is a way of responding to a fairly long-term change in the environment of the organism by producing a fairly long-term change in the pattern of its metabolism. 19 Finer forms of regulation, working inside cells, rather than changing flow rates through metabolic pathways by changing concentrations of enzymes (e.g. by changes in gene expression or zymogen activation), instead change flow rates by changing the activities of enzymes already present. 20 BI25M1 METABOLIC REGULATION LECTURE 2 AIM: To review: metabolic regulation involving changes in enzyme activity; roles of covalent modification and allosteric effectors in changing enzyme activity. [Lehninger Edition 4 pp. 21-22; 225-233; Chapter 15 Lehninger Edition 5 pp. 20; 220-227; Chapter 15 Instant Notes Sections C5, J7] 21 In Lecture 1, we saw that changes in flow rates through metabolic pathways could be brought about by changing the concentration of enzymes (through changes in gene expression or zymogen activation). This results in on/off switches; rather long-term changes in metabolic flow rates; and hence effective, but coarse systems of metabolic regulation. A finer form of regulation involves not synthesising or degrading the enzymes of a metabolic pathway faster or slower, but simply taking the enzyme molecules already present in the cell and making them work better (or worse) as catalysts. 22 This is done by making enzymes interact with something that affects their activity. It is potentially a finer form of regulation because it should: 1 be fast; 2 produce reversible changes (if the effect of whatever interacts with the enzyme is reversible); 3 allow fine tuning of metabolic flow rates, rather than just switching flow on or off (if changes in enzyme activity can be made to occur to various degrees). The two main ways in which changes in enzyme activity are brought about are: 1 covalent modifications; 2 allosteric effects. 23 CHANGES IN ENZYME ACTIVITY BROUGHT ABOUT BY COVALENT MODIFICATION [Enzymes Lecture 4] By changing one amino-acid in a polypeptide, a base-change (point) mutation can greatly alter the 3D structure of a protein, and hence alter its activity. An example is the glutamate to valine change in sickle-cell haemoglobin. [BI20M1 Genetic Code and Translation Lecture 1] Similarly, a temporary (rather than genetic) change in one amino-acid R group of an enzyme protein, should have the potential to alter the 3D structure of the enzyme, and hence alter its activity. 24 In fact, several kinds of reversible, covalent modifications of R groups of amino-acid monomers of enzyme proteins occur inside cells. Most common is phosphorylation of -OH groups. These are present in the R groups of three coded amino-acids: serine threonine tyrosine. In some enzymes, one or more of such amino-acid monomers can be phosphorylated, to produce phosphoserine phosphothreonine phosphotyrosine. 25 Phosphorylation changes the amino-acid monomer R group markedly, making it bulky and charged. [This is analogous to the marked structural change from the R group of glutamate to that of valine in sickle-cell haemoglobin.] The change alters the 3D structure of the enzyme, and hence its activity. [Similarly, the genetically-caused R group change alters the activity of the haemoglobin.] 26 The R group phosphorylations catalysed by kinases, and are reversed phosphatases. by the action are of [You have met many other kinases and phosphatases catalysing similar reactions in BI25M1.] ATP ADP enzyme-OH enzyme-OP kinase Pi enzyme-OH enzyme-OP phosphatase 27 For some enzymes, phosphorylation activates the enzyme; for others, it inhibits the enzyme. [Examples of both situations are in Lecture 3] A look back at the process: a change in enzyme activity is caused by a change in the 3D structure of the (already present) enzyme protein which itself is produced by covalent modification of an amino-acid R group. This is an effective, fine form of regulation, because: 1 it is fast (as it relies on enzyme-catalysed changes in protein structure); 28 2 it is reversible (through the co-ordinated activities of kinase and phosphatase); 3 it has the potential to allow fine tuning of metabolic flow rates, rather than just acting as an on/off switch (as phosphorylation and dephosphorylation can be made to occur to various degrees). This type of regulation is, therefore, a way of responding fast to changing circumstances. In a way, it resembles zymogen activation. There, as here, a pre-formed biomolecule is made to change its activity, but here, the change is not necessarily allor-nothing, and it is reversible. 29 CHANGES IN ENZYME ACTIVITY BROUGHT ABOUT BY ALLOSTERIC EFFECTS [Enzymes Lecture 4] Enzymes that are subject to allosteric effects usually 1 have quaternary structure; 2 show sigmoidal rather than hyperbolic (Michaelis-Menten) curves when Vo is plotted against [S]. 30 The curve shows that an increase in [S] at low [S], e.g. between a and b, produces a small increase in Vo, while the same increase at higher [S], e.g. between b and c, produces a greater increase in Vo. This suggests that the presence of low [S] somehow sensitises the enzyme, so that it responds to the increase in [S] at high [S] more efficiently. We saw two models [Enzymes Lecture 4: Concerted and Sequential models] that tried to explain the odd, sigmoidal curve. 31 Both models used the point allosterically affected enzymes quaternary structure that had to explain that binding of some substrate increased the efficiency with which the enzyme bound more substrate. [See Enzymes Lecture 4 for details of the models] 32 As well as being sensitive to changes in [S], the activity of allosterically affected enzymes is also influenced by other effectors. These allosteric effectors bind to a site on the enzyme other than the active site. [‘allo steric’ = ‘other shape/structure’] The binding is non-covalent. The binding either stabilises a particular 3D structure of the enzyme or causes the enzyme to a adopt a new, particular 3D structure. [The Concerted and Sequential models mentioned earlier explain what happens in these two different ways; see Enzymes Lecture 4.] 33 Whatever, the stabilised or adopted 3D form of the enzyme binds substrate differently. For allosteric activators, binding to the enzyme stabilises or induces adoption of a 3D form that binds substrate better, and hence the enzyme is activated. For allosteric inhibitors, binding to the enzyme stabilises or induces adoption of a 3D form that binds substrate worse, and hence the enzyme is inhibited. 34 Ordinary cell metabolites, e.g. G6P, ATP, at certain concentrations, become allosteric effectors for particular enzymes. Thus, in the feed-back inhibition example, [Lecture 1] at a particular concentration, isoleucine becomes an allosteric inhibitor of the first enzyme of its anabolic pathway. [Other examples of allosteric effectors are in Lecture 3] 35 A look back at the process: a change in enzyme activity is caused by a stabilisation of (or change in) the 3D structure of the (already present) enzyme protein which itself is produced by non-covalent binding of an effector to an enzyme site other than the active site. This is an effective, fine form of regulation, because: 1 it is fast (as it relies on binding of an effector to an enzyme); 2 it is reversible (as the binding is non-covalent); 36 3 it has the potential to allow fine tuning of metabolic flow rates, rather than just acting as an on/off switch (as the activation/inhibition can be made to occur to various degrees). As we have seen, allosteric effectors metabolites. are ordinary cell Small changes in metabolic flow patterns inside cells are detected as small increases in concentrations of these metabolites. At these concentrations, the metabolites then trigger counter-balancing flow changes elsewhere in the flow system by acting as allosteric activators or inhibitors. 37 The allosteric effector system, then, with its subtle detection and triggering processes, is a major factor in the maintenance of the delicate balance and economy of metabolism inside cells. 38 BI25M1 METABOLIC REGULATION LECTURE 3 AIM: To review: the control of glycogen metabolism, a process that illustrates several of the general points made in Lectures 1 and 2 [Lehninger Edition 4 pp. 21-22; 225-233; Chapter 15 Lehninger Edition 5 pp. 20; 220-227; Chapter 15 Instant Notes Sections C5, J7] 39 Glycogen metabolism is regulated by a system involving both covalent modifications and allosteric effects. Glycogen is a storage polysaccharide of skeletal muscle and liver. [Carbohydrates Lectures 1, 2) and Intermediary Metabolism It has different functions in these two sites. glycogen catabolism skeletal muscle G6P liver G6P glycolysis ATP (for contraction) Glc (to replenish blood glucose) 40 Glycogen synthase is a major enzyme of glycogen anabolism; glycogen phosphorylase is a major enzyme of glycogen catabolism. Both enzymes can be covalently modified by phosphorylation/dephosphorylation of serine R groups. [as discussed in Lecture 2] 41 ATP ADP glycogen phosphorylase b glycogen phosphorylase a phosphorylase kinase* normally inactive active Pi phosphatase *We see later that THIS enzyme can also exist in phosphorylated (active) and dephosphorylated (inactive) forms. 42 ATP ADP glycogen synthase a glycogen synthase b protein kinase A active normally inactive Pi phosphatase 43 The role of covalent modifications and allosteric effects in regulating glycogen metabolism can be illustrated by considering events in skeletal muscle when at rest during mild exercise when preparing for vigorous exercise. When at rest The non-phosphorylated forms of the two enzymes predominate. glycogen phosphorylase b (normally inactive) glycogen synthase (active) a Result: at rest, the muscle gradually stores Glc as glycogen. 44 During mild exercise Although glycogen phosphorylase b is normally inactive, its activity can be altered by allosteric effectors. ATP and G6P are allosteric inhibitors of glycogen phosphorylase b, and AMP is an activator. When the muscle is at rest, the balance of concentrations of the three effectors keeps glycogen phosphorylase b inactive. [as we’ve seen] 45 When the muscle is mildly exercised, [AMP] rises [ATP] and [G6P] fall and glycogen phosphorylase b is activated. Result: some glycogen is degraded to provide ATP for contraction. 46 When preparing for vigorous exercise With impending ‘fight or flight’, the hormone epinephrine is released by the adrenal medulla. epinephrine binds to -adrenergic receptor on membrane of muscle cell causes GTP to bind to membrane protein Gs Gs-GTP binds to and activates membrane adenylyl cyclase adenylyl cyclase catalyses ATP cAMP [‘cAMP’ is cyclic AMP: its structure is in Lehninger, Edition 4 p.302; Edition 5 p.298] cAMP allosterically activates protein kinase A 47 protein kinase A phosphorylates glycogen synthase (a b) i.e. inactivates it phosphorylates phosphorylase kinase i.e activates it [see earlier asterisk*] glycogen anabolism inhibited which phosphorylates glycogen phosphorylase (b a) i.e. activates it glycogen catabolism activated Result: glycogen is degraded to provide ATP for the contraction necessary in the stressful circumstances. 48 Why did such a complex system evolve? The way in which epinephrine triggers muscle glycogen catabolism is typical of many other processes in which signals arriving at a cell trigger some change in cell activity e.g. responses to other hormones and growth factors; perception of sight, smell, taste; control of the cell cycle. All such biosignalling processes involve the amplification of a signal through a cascade of events. 49 In the epinephrine/glycogen example, the signalling process involves a cascading sequence, in which a few molecules of an enzyme act on many other molecules of another enzyme which then act on very many molecules of yet another enzyme and so on, so that, at each step, the signal is amplified. In this way, a few hormone molecules are able to bring about the mobilisation of a very large number of glycogen molecules. 50 A related biosignalling cascade involves Ca2+ in the regulation of muscle glycogen metabolism. nerve impulse signalling contraction received by muscle cell causes release of Ca2+ from sarcoplasmic reticulum into cytosol Ca2+ activates the dephosphorylated form of phosphorylase kinase glycogen phosphorylase is phosphorylated (b a) i.e. activated glycogen catabolism activated Result: glycogen is degraded to provide ATP for contraction. 51 Relaxation processes In Lecture 1, we saw that, in general terms, for balance and economy in organisms to be achieved, any change in one part of a metabolic flow system must 1 be detected; 2 trigger a counter-balancing flow change elsewhere in the flow system. In hormonal and Ca2+ regulation of glycogen metabolism, the ‘change’ that needs to be detected is the energy requirement for muscle contraction, and the appropriate flow change (increased glycogen catabolism) is triggered through the processes seen. 52 Such regulation can only be effective if there is a way to shut down the triggered response once the need for it has passed. Thus, in our example, glycogen catabolism must not continue, once the energy needs that triggered it have been met. The triggered response must be ‘relaxed’. Relaxation processes in catabolism cascades are: the glycogen 1 Ca2+ is pumped back from cytosol to sarcoplasmic reticulum 2 epinephrine production stops 3 GTP is converted to GDP 53 4 cAMP is converted to AMP 5 phosphatase [seen earlier] catalyses dephosphorylation of phosphorylase kinase (inactivates) glycogen phosphorylase (a b) (inactivates) glycogen synthase (b a) (activates) Result: [as seen before] at rest, the muscle gradually stores Glc as glycogen.