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Metabolism BIOC2005 – Dawson Lecture notes L1 – Regulatory Strategies in Metabolism - - - Regulation is necessitated because organisms need to be able to respond to changes in environment in order to meet metabolic needs o E. energy needs, carbon source, nitrogen requirements, minerals needed for bi...

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Metabolism

BIOC2005 – Dawson Lecture notes L1 – Regulatory Strategies in Metabolism -

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Regulation is necessitated because organisms need to be able to respond to changes in environment in order to meet metabolic needs o E.g. energy needs, carbon source, nitrogen requirements, minerals needed for biosynthesis or as cofactors o Or changes in external conditions e.g. available carbon sources, solute concentration, pH changes, or an event like a muscle contraction Intracellular environment is extremely crowded o Hydrophilic substrates must be transported to other compartments by transporter proteins o Hydrophobic substrates associate with other components – most often membranes Cellular localisation (the location of substrates or enzymes within a cell) play a key role in regulating metabolism

Conserved Metabolites - Some substrates, namely coenzymes, are conserved – they may be altered and then regenerated, but the total concentration within the cell of both forms is unchanged (e.g. NADH/NAD) o More complex for ATP because there are 3 forms, cells work to keep [ATP] and [ADP] much higher than [AMP] to maintain concentrations

Substrate availability - Cells often regulate internal processes according to which substrates are available: o Alter transport proteins to take up different substances, or increase the amount of one protein made to take up more of a substrate o Halt uptake of other substances if better ones are available (e.g. the lac repressor if glucose is present)

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o Alter metabolic pathways to process different substrates o Synthesis substances not available from surroundings (e.g. making alanine from glutamine if no food available?) Note: in multicellular organisms like humans, extracellular environment is kept fairly constant (tissue fluid, blood plasma etc.) o This means that if fatty acids are needed at tissue x, they might be released at tissue y and transported o This can be further facilitated by removing transport proteins for substrate intake in tissue x, meaning more substrate can be spared for y

Protein Regulation - Can occur at many levels depending on how fast a response is needed, or for how long a response will likely need to last - Transcription o Unicellular organisms  Unicellular DNA codes for proteins to allow cell to cope with a range of conditions, and can control the transcription of these genes as required (i.e. up-regulation of rus operon if Fe2+ is present in A. ferrooxidans) o Multicellular organisms  All cells contain the same DNA (with some exceptions), and cells behave very differently – transcriptional control is the reason for this, and is the cause of most long-term metabolic regulation - mRNA Stability o Alter the rate at which mRNA is degraded, some sequences may have stemloop structures to make them harder for nucleases to recognise, and as such more readily translated when needed  This is a faster response than transcriptional control - Translation o Alter the rate at which mRNA is translated into protein - Degradation/ubiquitination o Tag proteins with ubiquitin to target them for degradation in 26S proteasomes – this permanently inactivates an enzyme and can be used to downregulate certain pathways - Turnover o Turnover is the combination of transcription, translation, degradation all happening consecutively giving rise to unique half-life values for proteins. These essentially serve as a measure of the stability of the protein o Lower half-life means a higher rate of synthesis + degradation, so low half-life proteins can increase in concentration rapidly by halting degradation - Location o Proteins must be moved to the correct location within a cell to carry out their function, this effect can take seconds or minutes

E.g. GLUT4 is a glucose transporter, and is directed towards the membrane in the presence of insulin to stimulate glucose uptake and lower blood glucose levels  E.g. glucokinase is stored into the nucleus until it is required for glycolysis o The reason for this storage is that synthesising these proteins requires ATP and it may not even be used, so localising and transporting them this way is less wasteful for the cell Allosteric Control o Regards the reversible binding of allosteric ‘effectors’ to an enzyme, away from the active state. The effects of this can be seen within seconds 

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o Some allosteric proteins are oligomeric e.g. haemoglobin, where substrate binds the R-state of one subunit and stabilises the R state of other subunits, giving rise to a sigmoidal relationship of rate vs. [S]

o In the case of haemoglobin, this allows for dissociation of all of its oxygen over a very small range of ppO2, increasing its efficiency for oxygen transfer to respiring tissues o Competitive inhibition increases Km (a higher [S] is required to reach 0.5 Vmax) and can do this by binding enzyme or substrate o Noncompetitive inhibition decreases Vmax (binds enzyme and E:S complex, which functionally decreases [E])

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o If the allosteric effector is the enzyme’s substrate the effect is called homotropic. Otherwise, the effect is heterotopic o In the above diagram, + from one pathway activates the enzyme in the central pathway. Working against this is feedback inhibition, where too much of the – substrate on the left is toxic, and alters another enzyme in the pathway to decrease the rate of the process.  Feedforward activation means the triangle activates the enzyme to stop blockage if the end-product is at safe concentrations within the cell Covalent modification o Covalent modification is a very stable way (lasts minutes to hours) or altering protein behaviour  E.g. phosphorylation (namely of -OH in Ser, Thr and Tyr residues)  Not as quick as allosteric control but still very useful o This can give rise to reaction cascades, used in signal transduction pathways – meaning many proteins can have their behaviour altered by a very small number of different hormones  E.g. Protein kinases are activated, transfer the phosphate to another protein and inactivate themselves  Phosphorylation can activate or inhibit proteins Regulatory proteins o Many enzymes in a pathway function close to equilibrium – they can catalyse the forward or backward reaction.

Some of these enzymes catalyse FAR from the equilibrium, and these are called rate-limiting enzymes  These reactions are exothermic, and have extremely negative changes in the Gibbs energy, so are irreversible o These irreversible steps are often the first ‘unique’ or ‘committed’ step, where the energy cost of returning a substrate to its previous state would be enormous o Regulatory enzymes may also respond to allosteric control and covalent modification 

o Flux control coefficient is experimentally determined by seeing the effect of changing enzyme concentration on the concentration of the two possible branched pathways here  This can be used to see how gene regulation alters the output of metabolic pathways

Lecture 2 – Fatty Acid Biosynthesis -

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Roles of Fatty Acids

o Note, phospholipids often contain unsaturated FAs. Signalling here often means as hormones Important of FA Synthesis o In mammals, occurs almost exclusively in the fed state  In the liver where glycogen stores are full, excess glucose is used to make palmitic acid -> TAG -> VLDL -> blood  In adipose, glucose is used for making palmitic acid -> TAG -> stored as fatty droplets  In lactating mammary gland glucose makes shorter FAs than palmitic acid since newborns cannot properly metabolise longer FAs  In foetuses FA synthesis supplies FAs for brain development and lung surfactant  In typical Western diets, high fat content suppresses FA synthesis

Overview of FA Synthesis:

o Pyruvate -> Acetyl CoA via pyruvate dehydrogenase in the mitochondria o Not all of the acetyl CoA goes through the Krebs cycle (Citric Acid Cycle), some is phosphorylated to malonyl CoA and made into palmitic acid using NADPH o Palmitic acid can then be used for FA oxidation, or the uses outline previously

o FA Oxidation uses many enzymes for different lengths of FAs  FA synthesis uses FA synthase which is multifunctional and works on many lengths of FA o FA Synthesis occurs in fewer tissues than FA Oxidation, need a large amount of NADPH to reduce FAs, NADPH isn’t found in high concentrations in all tissues  NB, malonyl CoA is more reactive than acetyl CoA

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How does Acetyl CoA enter cytosol?

o Normally in the Krebs cycle, oxaloacetate -> citrate via citrate synthase. But, if aconitase is saturated – citrate leaves the cytosol  This happens at high citric acid cycle activity, citrate signals that more fuel is being broken down o Then in the cytosol, citrate -> oxaloacetate via citrate lyase, and in the process CoASH is made into Acetyl CoA using water and one ATP. From here, Acetyl CoA can be made into malonyl CoA via acetyl CoA carboxylase (a regulated enzyme). And then malonyl CoA can go on to be synthesised up to palmitic acid. o This happens only in the fed state for liver and adipose

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Sources for the required NADPH

o If citrate is removed, oxaloacetate in the CAC is not regenerated  Cytosolic malate dehydrogenase regenerates oxaloacetate via malic enzyme using the reducing power of NADH  The resulting NAD can re-enter glycolysis  Malate is oxidised to pyruvate by oxidised NADP+, regenerating an NADPH and allowing regeneration of oxaloacetate o Malic enzyme provides one NADPH per acetyl CoA transported into the cytosol  Synthesis of palmitic acid requires 8x acetyl CoA and 14x NADPH  So, the remaining 6 NADPH can come from the pentose phosphate pathway, requiring 3 x glucose:

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Fatty Acid Synthase o In higher animals, FA Synthase is a homodimer with 7 domains (7 monomers twister around each other)  In lower animals, these 7 domains are separate enzymes

Fatty Acid Synthesis -

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o Can see malonyl CoA forms a thiol bond with the ACP domain o Malonyl CoA splits and the fragment binds elsewhere on the enzyme o CO2 is released as the chain length increases  Reduction, dehydration and another reduction follows o The chain length increases and the cycle repeats o When chain is 16 carbons long, the FA is desaturated or elongated in the SER FA synthase occurs as a homodimer, but functions even if one monomer doesn’t function

o The ACP (acyl carrier protein) has a phosphopantetheine side arm, this is the same as the end of acyl CoA, and is highly flexible. FAs attach to the free -SH group here and can move to the keto-acyl synthase (KS) domain when malonyl CoA comes in, where it attaches to the -SH on a Cys residue o AT is acyl transferase, takes acyl from acetyl CoA or malonyl CoA and transfers to KS  After this, another malonyl joins the growing chain at the ACP o Malonyl CoA has a -COOH group, decarboxylation allows the C to move from KS to ACP o C=O must be reduced to CH2 o DH is dehydratase o C is added to the ‘right-hand side’ of growing FA Chain -

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Fatty Acid Modifications o i.e. enzymes have a ‘preference’ for certain FAs, and might be less specific than other FAs  Palmitic not preferred over other FAs necessarily o FAs also have structural roles requiring specific lengths/double bonds of correct configurations o Essential FAs cannot be synthesised – must come from diet and then undergo modification o Double bonds in phospholipid FAs increase fluidity of membranes by introducing kinks, improving the mobility of membrane components  Pi bonds give ‘kinks’ and increase disorder, preventing crystallisation of some membrane regions o There is some (though poorly understood) tissue-specific regulation of FA modification n-6 series o Made by terrestrial plants o Most common is linoleic acid (18:2n-6) o Most important for humans is arachidonic acid (20:4n-6)  An important FA for inositol phosphoglycerides, and some other phospholipid types  Also can be cleaved from phospholipids when stimulated by cytokine/hormone and converted into signalling molecules called eicosanoids for signalling cascades

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n-3 series o Made by cold-water plants (mostly α-linolenic acid, 18:3n-3) o Modified by fish who eat them, so humans get a mixture of DHA (22:6n-3) and EPA (20:5n-3) in their diet  DHA are highly enriched in phospholipids for brain and retina, very flexible and facilitate membrane-embedded proteins  EPA is anti-thrombotic and anti-inflammatory, competes with arachidonic acid for incorporation into phospholipids

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Polyunsaturated Fatty Acids (PUFAs) o Involved in gene regulation o Repress lipogenic enzymes, and upregulate gene expression favouring lipid oxidation o Bind specific nuclear receptors, which bind to response elements on DNA to enhance transcription  E.g. PPARs  When they bind their ligand they dimerize with RXR and bind ‘PPREs’ (RE meaning Response Element) to increase FA oxidation and uptake, decreasing proinflammatory cytokine production  So, inhibition of lipgenesis is PPAR-independent

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Natural Fatty Acids and Naming Conventions

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o Most FAs have even number of carbons o All of the above have 20 carbons and 4 double bonds o The Δ numbering system treats the C in COO as C1, the first double bond occurs at carbon 5, and there is a methylene interruption between each double bond o Miller and w don’t show the location of bonds, and number from the ‘unchanged end’, since increasing the chain length adds onto the COO end -

Elongation of FAs

o Takes place on Smooth ER because FAs are hydrophobic, and catalysed by FA elongases o Important because we need longer PUFAs for health and storage  E.g. most arachidonic acid (20:4n-6) is made from linoleic acid (18:2n6) in our diet o Elongation also allows storage of excess fatty acid, which can be shortened later  E.g. most arachidonic is accumulated as adrenic acid (22:4n-6) which can be shortened later if necessary -

Desaturation of fatty acids o Introduce pi bonds into the FAs, using electrons from cytochrome b5 to desaturate:

o Hu man cells have three

different desaturases, which desaturate at specific points:

o E.g. Δ9 adds a double bond between carbons 9 and 10, where carbon 1 is the COO carbon.

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Retroconversion of Fatty Acids o Retroconversion is the removal of two carbons (as acetyl CoA) from the α end of the FA, occurs in peroxisomes o This allows the cell store excess FAs as 2+ versions and cut them back down to a useful size later o Similar to β-oxidation in mitochondria, but here the FAD is regenerated using oxygen, producing hydrogen peroxide, which is broken down by catalase. The resulting acetylCoA and NADH are exported from the peroxisome

o Note, FAD here is not the same as in succinate dehydrogenase, because this is in the peroxisome.

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Fatty Acyl CoA + Glycerol

o Formation of triacylglycerol, which is how fat is used as an energy store

o TAG has a much higher energy yield and lower cost than glycogen, while taking up less space (glycogen is stored as random branches, while TAG is highly organised)  However, FAs cannot be converted into glucose, hence the importance of glycogen -

FA Synthesis and Dysregulation o As said above, Western diets are high in fat so inhibit fatty acid synthesis  FA Synthesis usually occurs in fed state at high [glucose] o Acetyl CoA Carboxylase produces malonyl CoA which inhibits FA oxidation and is the precursor for FA synthesis – this is the main point in FA metabolism’s regulation o FA synthesis is very low in people with severe diabetes due to low insulin concentration o In glucose-6-phosphatase deficiency (von Gurke’s disease) FA synthesis is very high, since glucose-6-phosphate is trapped in the liver

Nitrogen Metabolism -

Overview

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o Lots of unreactive N2 in the atmosphere o Diazotrophic bacteria fix nitrogen unto usable ammonium ions (NH4+), then other bacteria convert there into nitrates, into nitrites, back into ammonium and into organic N (i.e. amino acids used in protein) Nitrogen Fixation

o o Very negative delta G, but a high activation energy barrier since a triple bond must be broken o In the Haber process, high temperature and pressure is used to overcome this barrier o In biological process, a high energy input of 16 x ATP is needed to give the resulting 2 ammonia molecules, via an enzyme complex called the Nitrogenase complex

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The Nitrogenase Complex o Hydrolysis and binding of 2 x ATP enables reductase to donate an electron to nitrogenase

o Complex doesn’t work if oxygen is present o MoFe protein functions similar to FeS cluster o Fe proteins have ATP binding sites, so ATP can bind and change the conformation of the protein o Upon conformational change, ferredoxin is reduced and passes its electron to MoFe to nitrogen, at which point protons can join to form ammonia -

Incorporation into organic compounds

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Glutamate and Glutamine

o 3 is reversible, mostly used to remove ammonium o 2 shows alpha-ketoglutrate + glutamine being used to make 2 glutamate molecules o 3 shows glutamate being used to make alpha-ketoglutarate and release ammonium Transamination o The transfer of amino groups between keto-acids o Most aminotransferases are specific for glutamate and one other amino acid

o The reactions are easily reversible (Gibbs energy roughly 0, eq. constant roughly 1)

o Allows the nitrogen in glutamate to be used to synthesise other amino acids o In mammals, acts to funnel nitrogen into glutamate before disposal (see 3 above) o Vitamin B6 (pyridoxal phosphate) is vital cofactor for aminotransferases

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Pyridoxal Phosphate

o Schiff’s base results from adding C=O to NH2 giving the pictured bond o Pyridoxal phosphate then reacts with incoming amino acid to dissociate from Lys.  Use of another alpha-keto acid can drive backwards reactions

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Essential Amino Acids o Essential amino acids are only obtained through diet, and cannot be naturally synthesised by humans: o

o Arginine is ‘conditionally essential’, at some stages of growth we need more than we can make, so must eat to supplement this need.  Amino acids supply most of the nitrogen needed for nucleic acids, porphyrins, neurotransmitters etc.

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Nitrogen Metabolism in Mammals o Mammals take in more nitrogen than they need, so need to excrete it safely in a way that removes N but stores or makes use of (oxidises) the carbon skeleton o If an organism is growing, nitrogen balance is positive (more nitrogen is retained than excreted)  This is because a growing organism needs nitrogen to make proteins etc. o Mammals excrete N as urea to dilute nitrogen, urea is highly soluble and nontoxic o Aqueous animals excrete ammonium, but animals that need to save water or carry less water excrete uric acid  Uric acid is insoluble and reduces the required water intake o In humans, N is transported to the liver in the form of Glu or Ala, then transferred to Asp or ammonium and converted to urea in the urea cycle

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Channelling N into Urea

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o The liver here takes up ammonium from the blood, from Gln, from Glu, or from Asp (which also comes from Glu)

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The Urea Cycle

o First step is ammonium -> carbamoyl phosphate, which is made into citrulline o Citrullin then enters the cytosol and joins with Asp to make Argininosuccinate. o Argininosuccinate is metabolised into Arg, and then into Ornithine o Ornithine re-enters the mitochondria and the cycle can repeat  ...