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1 BI25M1 HOW ORGANISMS HANDLE NITROGEN LECTURE 1 AIM: To review: N-containing biomolecules; N flow into and out of the biosphere. [Lehninger Instant Notes Chapters 18, 22 Section M] 2 N-CONTAINING BIOMOLECULES The main ones are: 1 Proteins (contain amino-acids) 2 Nucleic acids and nucleotides (contain bases) 3 Active amines CO2 amino-acid 1 /2O2 + NH4 amine aldehyde ACTIVE INACTIVE (several, non-coded) INACTIVE 3 eg histamine serotonin dopamine (nor-)epinephrine. 4 Haem of haemoglobin and cytochromes. 4 N FLOW INTO AND OUT OF THE BIOSPHERE N in biomolecules comes ultimately from N2 (80% of the atmosphere). However, N2 isn’t readily exchanged with N found in organisms. (Compare with ready exchange between biological and non-biological C, H, O.) This is because N2 is very unreactive: N2 is N N. Only a few micro-organisms can reduce N2 and ‘fix’ it in the biosphere. Once ‘fixed’, as NH4+, N flows to other biomolecules through the (coded) aminoacid glutamate. Only a few (different) micro-organisms can pass N back to the atmosphere, as N2. 5 ATMOSPHERE N2 BIOSPHERE + NH4 glutamate (other) amino-acids proteins and other N-containing biomolecules amino-acids glutamate + NH4 N2 6 FLOW OF N FROM N2 INTO THE BIOSPHERE This is ‘N fixation’. It occurs in: no eukaryotes; a few prokaryotes, including Cyanobacteria; a few free-living bacteria; Rhizobium bacteroids living symbiotically in root cells of legumes. It is catalysed by nitrogenase, a complex enzyme containing Mo or V. Much ATP is needed (16 mol for each mol N2 reduced), because N N is so stable. 7 [Compare with industrial (‘Haber process’) reduction of N2 to +NH4, to make agricultural fertilizer, which needs: an iron catalyst, 5000C and 300 atmospheres.] There is much interest in the possibility of genetically engineering genes coding for the N fixation apparatus (including those encoding nitrogenase) into non-legumes, as biologically available N is often rate-limiting in crop development. 8 We do not know when N fixation evolved. Nitrogenase is strongly inhibited by O2. This suggests that it evolved before appearance of atmospheric O2. However, at that time, the atmosphere probably contained much NH3, so why N fixation would have been needed then is unclear. Whatever, present-day N fixers cope inhibition in several ways: with the some live anaerobically; some switch off nitrogenase synthesis when O2 is present; some uncouple mitochondria [TRS and Ox Phos Lecture 2]: this increases electron flow and O2 in cells rapidly ‘burns off’; 9 Cyanobacteria have specialised N-fixing cells (‘heterocysts’) with O2-inpenetrable walls; legumes produce leghaemoglobin, which, like RBC haemoglobin, binds O2 (and prevents the inhibition). These few, N-fixing organisms are responsible for virtually all the N flowing from atmospheric N2 towards the Ncontaining biomolecules made by organisms. 10 ROLE OF NO2- AND NO3- IN THE FLOW OF N FROM +NH4 INTO ORGANISMS N, now fixed as +NH4, is still not available to most organisms. It is converted by soil bacteria via NO2- (nitrite) into NO3- (nitrate). This process is called ‘nitrification’. As NO3-, it can now be taken up by many microorganisms and plants. Once inside these organisms, NO3- is converted back, through NO2-, to +NH4. 11 FLOW OF N FROM +NH4 TO OTHER BIOMOLECULES OCCURS VIA GLUTAMATE (Glu) The crucial reaction is: NAD(P)H + H+ + NH4 + -ketoglutarate NAD(P)+ Glu + (KG) COO- COO- (CH2)2 (CH2)2 C O COO- HC NH2 COO- [Glu is a coded amino-acid. KG is a citric acid cycle intermediate.] H2O 12 The reaction: can proceed in either direction (depending on the conditions); is an oxidative deamination when it proceeds R L; can use either NAD or NADP; is catalysed by (named for the R glutamate dehydrogenase L reaction); is crucial to life: Glu is the ONLY amino-acid that can obtain its N directly from +NH4 (in the L R reaction). 13 Once Glu is made, other amino-acids are made from it, (by transamination, as we see in Lecture 2), and, from them, other N-containing biomolecules are made. 14 FLOW OF N FROM THE BIOSPHERE BACK TO N2 Eventually, N excreted by organisms and from dead organisms becomes +NH4. The glutamate dehydrogenase reaction just seen is again important. Working in the R L direction + (that is, Glu NH4), it is a major way in which N from organisms flows to +NH4. Soil bacteria then convert +NH4, via NO2-and NO3-, back to N2. 15 Thus there is a very narrow organismal window, involving just a few micro-organisms, by which N enters and leaves the biosphere. The window is narrow biochemically, too, involving, as we’ve seen, crucial reactions of N2, + NH4, NO2-, NO3and Glu. 16 BI 25M1 HOW ORGANISMS HANDLE NITROGEN LECTURE 2 AIM: To review: N flow through the body; digestion of dietary protein; role of glutamate in transfer of N to, from and between amino-acids. [Lehninger Instant Notes Chapters 18, 22 Section M] 17 OVERVIEW OF N FLOW THROUGH THE BODY The main flow involves: 1 digestion of dietary protein; ‘turn-over’ of body protein; excretion of N as urea; 2 a pool of intra- and extra-cellular free amino-acids (kept constant at about 100g). 18 body protein dietary protein synthesis of some (‘non-essential’) amino-acids amino-acid pool body protein synthesis of other N-containing molecules N excreted as urea C or ATP glucose (gluconeogenesis) fatty acids ketone bodies CO2 19 DIETARY PROTEIN Dietary protein replenishes amino-acids used in anabolism and catabolism. (as shown in the previous diagram) Normally, N flow into and out of the body is balanced. N flowin that is less than N flowout results in ‘negative nitrogen balance’. This happens in the condition kwashiorkor. [‘Sickness of the older child when the next baby is born’] It occurs when carbohydrate intake is adequate, but N intake is poor, as sometimes happens when a child is weaned onto a starchy diet. 20 Among several consequences: low N intake low plasma albumin low plasma osmotic pressure lower than usual entry of interstitial water into plasma oedema plump belly N flowin that is greater than N flowout results in ‘positive nitrogen balance’. This occurs during rapid tissue growth: eg in children; pregnancy; recovery from illness; body-building. N taken in in excess of requirements cannot be stored (unlike carbohydrate, fat) and is excreted. 21 Of the 20 coded amino-acids, 10 (‘non-essential’) can be synthesised in the body (they have simple R groups); 8 (‘essential’) cannot be synthesised and must be present in the diet; 2 (arginine, histidine) can usually be made at adequate rates, but are needed in the diet when tissue growth is rapid. Animal protein is usually rich in all essential amino-acids. An exception is collagen (in gelatin). Particular plant proteins are deficient in some essential amino-acids. Vegetarians need to use a mix of protein sources. DIGESTION OF DIETARY PROTEIN 22 1 Stomach HCl denatures proteins and makes them accessible to degradative enzymes. The zymogen pepsinogen is cleaved to pepsin autocatalytically, and, later, by pepsin itself. Pepsin cleaves proteins to small polypeptides. 2 Small intestine Mucosal cell-surface enteropeptidase cleaves trypsinogen (secreted by the pancreas) to trypsin. Trypsin cleaves other pancreatic zymogens to elastase chymotrypsin; carboxypeptidases A and B. 23 These enzymes have different specificities: they cleave adjacent to different amino-acids. Together, they break polypeptides to free amino-acids and short peptides. In addition, a mucosal cell-surface aminopeptidase removes amino-acids one at a time from N termini. Amino-acids and short peptides are absorbed into mucosal cells by several methods, including ATP-driven Na+-dependent transport like that used for Glc. [Carbohydrates and Intermediary Metabolism Lecture 2] Absorbed peptides are broken to amino-acids by a cytosolic peptidase. Amino-acids move through the portal system to the liver and are either metabolised directly or released into the general circulation. 24 body protein dietary protein synthesis of some (‘non-essential’) amino-acids amino-acid pool body protein synthesis of other N-containing molecules N excreted as urea C or ATP glucose (gluconeogenesis) fatty acids ketone bodies CO2 25 ROLE OF GLUTAMATE IN THE TRANSFER OF N TO, FROM AND BETWEEN AMINOACIDS Glutamate (Glu), one of the 20 coded amino-acids, forms a link in the flow of N between +NH4 and other amino-acids (as we saw in Lecture 1), and from one amino-acid to another. It does this by taking part in 2 reactions: 26 1 oxidative deamination (as seen in Lecture 1) NAD(P)H + H+ + NH4 + -ketoglutarate NAD(P)+ Glu + (KG) COO- COO- (CH2)2 (CH2)2 C O COO- HC NH2 COO- catalysed by glutamate dehydrogenase. H2O 27 All others obtain their N from a pre-existing amino-acid (as in the following reaction, mentioned in Lecture 1). 2 transamination (aminotransfer) Glu eg + -ketoacid pyruvate CH3 C O COO- -KG + amino-acid alanine CH3 HC NH2 COO- and oxaloacetate aspartate COO- COO- CH2 CH2 C O COO- HC NH2 COO- 28 These reactions: can proceed in either direction (depending on the conditions); are catalysed by transaminases (aminotransferases); use pyridoxal phosphate (made from vitamin B6) as a cofactor. Measurement of (normally intracellular) transaminases in plasma allows progression of liver and heart disease (in which cell damage and enzyme leakage occur) to be followed. 29 In summary: Glu, through these two reactions, plays a central role in: 1 movement of N from +NH4 to amino-acids + NH4 KG amino-acid Glu -ketoacid reaction 1 2 reaction 2 movement of N from amino-acids to +NH4 amino-acid KG -ketoacid Glu reaction 2 + NH4 reaction 1 30 3 transfer of N from one amino-acid to another amino-acid 1 KG amino-acid 2 -ketoacid 1 Glu -ketoacid 2 reaction 2 reaction 2 31 BI25M1 HOW ORGANISMS HANDLE NITROGEN LECTURE 3 AIM: To review: catabolism of body protein; transport of N to the liver; formation of urea for excretion. [Lehninger Instant Notes Chapters 18, 22 Section M] 32 CATABOLISM OF BODY PROTEIN This is part of the overview of N flow through the body that we saw in Lecture 1: body protein dietary protein synthesis of some (‘non-essential’) amino-acids amino-acid pool body protein synthesis of other N-containing molecules N excreted as urea C or glucose (gluconeogenesis) fatty acids ketone bodies ATP CO2 33 OVERVIEW OF THE FLOW OF N FROM CATABOLISED PROTEIN TO UREA body protein 1 amino-acids 2 Glu 3 + NH4 4 glutamine transported through plasma to liver 5 + NH4 + Glu 6 or 7 aspartate + NH4 urea transported through plasma to kidney for excretion 34 Also, in skeletal muscle: 2 Glu 8 alanine transported through plasma to liver 9 Glu 10 aspartate or + 11 NH4 35 STEPS IN THE FLOW OF N FROM CATABOLISED PROTEIN TO UREA 1 300-400 g body protein is ‘turned-over’ (and replaced) daily. It is broken to amino-acids by intracellular proteinases. 2 Amino-acid -amino group N transferred to Glu by transamination. (as seen in Lecture 2) amino-acid + KG N Glu + -ketoacid N 3 Glu is oxidatively deaminated. (as seen in Lectures 1 and 2) Glu N KG + + NH4 N is 36 4 Ammonia is very toxic. Hyperammonaemia (high plasma [+NH4]) causes tremors; speech slurring; coma; death. Probably, high [+NH4] pulls the oxidative deamination reaction [Step 3] towards Glu synthesis KG, a component of the citric acid cycle, is depleted ATP production by oxidative phosphorylation decreases brain is vulnerable to low [ATP]. 37 Also, perhaps high [+NH4] depletes ATP by excessive flow through the glutamine synthetase reaction [see below]. And Glu and its derivative -aminobutyrate are concerned with neurotransmission: perhaps changes in their concentrations when [+NH4] is high also contribute to the symptoms. Because of ammonia toxicity, N is transported from many body tissues through plasma as non-toxic glutamine [one of the 20 coded amino-acids]. ATP + NH4 N ADP + Pi glutamine synthetase glutamine 2N Glu N COO(CH2)2 CONH2 (CH2)2 38 5 In the liver, glutamine is converted back to + NH4 and Glu. H 2O + glutamine NH4 + Glu N N glutaminase 2N 6 Glu now either passes its N to aspartate by transamination (as seen in Lecture 2), Glu + oxaloacetate N KG + aspartate N 7 or is oxidatively deaminated [as in step 3]. Glu N KG + + NH4 N 39 The N of the +NH4 and aspartate formed in the liver in steps 5-7 becomes N of urea. Skeletal muscle can also transport N from its degraded proteins through plasma as alanine. [See the ‘Overview’ diagram] 8 Glu, instead of being oxidatively deaminated [as in step 3], is transaminated to alanine [as seen in Lecture 2]. Glu N + pyruvate KG + alanine N Non-toxic alanine is now transported through plasma. 40 9 In the liver, alanine is transaminated back to Glu. [reverse of step 8] 10 Glu is then either transaminated to aspartate. [as in step 6] 11 or oxidatively deaminated to give +NH4. [as in step 7] As before, the N of the +NH4 and aspartate formed in the liver in steps 10 and 11 becomes N of urea. 41 Why transport N as glutamine or alanine, rather than Glu? Glu has a net negative charge: its transport would mean additional transport of a cation; also, its charge means it does not readily pass through membranes. Alanine and glutamine bear no net charge. [The reason why alanine in particular is transported from skeletal muscle is discussed in Lecture 4.] 42 FORMATION OF UREA FOR EXCRETION N from catabolised protein is now in the form of aspartate and +NH4 in the liver. It is transformed into N of urea. 43 The urea cycle is shown in Lehninger, Edition 4 p. 666; Edition 5 p.683; Instant Notes, p. 409. 44 To make it simpler: 2N (as urea) 1N (from +NH4 via carbamoyl phosphate) 2N 4N 3N 4N 1N (from aspartate) 45 Another version: [1N] [1C] + NH4 + CO2 2ATP 2ADP + Pi NH2 CO O OPO O H2N C O H2N urea carbamoyl phosphate [2N, 1C] ornithine arginine citrulline aspartate [1N] fumarate ATP AMP + PPi arginosuccinate 46 Arginine is one of the 20 coded amino-acids; ornithine and citrulline are uncoded aminoacids. Carbamoyl phosphate and citrulline syntheses occur in the mitochondrial matrix; the other reactions are cytosolic. Note the energy expended in excreting N as urea. 47 Returning to hyperammonaemia: ‘Acquired hyperammonaemia’ may occur during liver malfunction, as in cirrhosis caused by alcoholism; hepatitis; bile duct obstruction. In such conditions, blood supply to the liver is impaired, metabolism of N in the liver is prevented, and [plasma +NH4] increases. ‘Hereditary hyperammonaemia’ occurs when one or other of the urea cycle enzymes is genetically deficient. 48 Treatment includes: 1 lowering (but not eliminating) protein intake, to reduce load on the urea cycle. 2 iv benzoate glycine iv phenylbutyrate Gln Glycine and Gln synthesis, to replenish that removed, helps reduce [plasma +NH4]. non-toxic, readily excreted products 49 3 Partial deficiency in a urea cycle enzyme may be treated by specific supplementations /limitations in diet. Total deficiency of an enzyme is likely to be fatal. Kidney failure can hyperammonaemia. also result in A little liver urea passes (quite normally) to the gut lumen. Most is broken to +NH4 by bacteria. Some of this is lost in faeces; some moves into plasma. In kidney failure, more urea than usual passes to the gut, and more than usual +NH4 produced there moves into plasma. Treatment: oral antibiotics to inhibit bacterial synthesis. + NH4 50 WHY EXCRETE N AS UREA? Urea is water-soluble and non-toxic. Prokaryotes, unicellular eukaryotes, bony fish, surrounded by water, excrete N as +NH4. It is quickly diluted and thus harmless. Excretion of (water-soluble) urea means that much water needs to be carried and eliminated: birds (carrying lots of water is awkward for flight) and reptiles (tend to live in arid areas) excrete N as relatively insoluble uric acid. 51 BI25M1 HOW ORGANISMS HANDLE NITROGEN LECTURE 4 AIM: To review: fate of the C of catabolised body protein; why alanine is transported from muscle; phenylketonuria. [Lehninger Instant Notes Chapters 18, 22 Section M] 52 FATE OF THE C OF CATABOLISED BODY PROTEIN Refer back to the overview of N flow through the body that we saw in Lecture 2: body protein dietary protein synthesis of some (‘non-essential’) amino-acids amino-acid pool body protein synthesis of other N-containing molecules N excreted as urea C or glucose (gluconeogenesis) fatty acids ketone bodies ATP CO2 53 and recollect the opening steps of the flow of N from catabolised protein to urea that we saw in Lecture 3. They were: body protein 1 amino-acids 2 2 Glu 3 + NH4 Glu or, in skeletal muscle, 8 alanine 54 Step 2 was the transamination: amino-acid + KG N Glu + -ketoacid N This can also be represented thus: KG amino-acid N and C Glu N -ketoacid C We traced the flow of N (eventually to urea). What happens to the C? 55 C of amino-acids is converted into the C of intermediates of: glycolysis/gluconeogenesis; citric acid cycle; lipid metabolism pathways. Sometimes the conversion is simple: eg the C of alanine and aspartate, by transamination, becomes the C of pyruvate and oxaloacetate respectively [as we saw in Lecture 2]. For other amino-acids, the conversion involves several steps. 56 To summarise, the flow for the various C atoms of the various amino-acids is: phosphoenol pyruvate pyruvate acetyl CoA oxaloacetate acetoacetate citrate fumarate succinylCoA KG 57 Any of these flows allows C atoms to be metabolised to CO2 through terminal respiration, providing ATP by oxidative phosphorylation. Those C atoms that flow to pyruvate or citric acid cycle intermediates [unfilled arrows in the diagram] can also generate Glc by gluconeogenesis. This occurs at a high rate during starvation, when glycogen (and triacylglycerol) stores are depleted, and muscle protein is degraded to provide Glc . [Citric Acid Cycle Lecture 2; Metabolic Retrospective lectures] 58 [Remember ‘feed-in’ or ‘anaplerotic’ reactions from Citric Acid Cycle Lecture 2, when a citric acid cycle intermediate is produced, as in the scheme above, the ‘feed-in’ reaction allows a section of the cycle to become part of a linear pathway, and contribute to anabolism (here, of Glc).] In contrast, those C atoms that flow to acetyl CoA or acetoacetate [filled arrows in the diagram], can flow to fatty acids and (other) ketone bodies, but are not gluconeogenic, for the reasons given in Citric Acid Cycle Lecture 2: pyruvate dehydrogenase-catalysed irreversible; reaction is there is no net oxaloacetate synthesis through the citric acid cycle. 59 Amino-acids providing pyruvate or citric acid cycle intermediate C atoms are therefore called ‘glucogenic’. Those providing acetyl CoA or acetoacetate C atoms are called ‘ketogenic’. Some amino-acids are both glucogenic and ketogenic (their different C atoms flow to different places). 60 WHY DOES SKELETAL MUSCLE TRANSPORT N FROM CATABOLISED PROTEIN AS ALANINE? Recollect from Lecture 3 that other tissues transport the N as glutamine, but that muscle, in addition, can transport N as alanine. most body tissues working skeletal muscle body protein 1 amino-acids 2 2 Glu Glu 3 + 8 NH4 alanine 4 glutamine transported through plasma to liver 61 Step 8 in the scheme above is the transamination: Glu N + KG + pyruvate alanine N We traced the flow of the N (eventually to urea). But the alanine also carries C from the skeletal muscle to the liver. The C comes pyruvate. from skeletal muscle [see reaction above] In the liver, the transported alanine is transaminated back to Glu and pyruvate. [Step 9 in Lecture 3] 62 Thus, pyruvate, generated by glycolysis in working skeletal muscle, is transported to liver (in the form of alanine), where it can be used in gluconeogenesis. This process is similar to, and backs up, the Cori cycle, in which lactate is moved from working skeletal muscle to the liver. [Carbohydrates and Intermediary Metabolism Lecture 4] Thus alanine transport simultaneously provides safe movement of N from catabolised muscle protein, and movement of pyruvate C for regeneration to Glc. 63 PHENYLKETONURIA Normally, phenylalanine (Phe) (one of the 20 coded amino-acids), is hydroxylated to tyrosine (another of the 20). A defect in the enzyme responsible leads to increased [Phe and its metabolites] in plasma and urine. Untreated, this leads to mental retardation, perhaps because the high [Phe] competes with movement of other aminoacids across the blood-brain barrier. Neonates are routinely screened for plasma Phe. 64 Treatment: restriction of dietary Phe (it is one of the ‘essential’ amino-acids) [Lecture 2] to amounts required for protein synthesis. Non-sugar sweeteners e.g. Silver Spoon, Hermesetas, Canderel, and some ‘diet’ soft drinks contain the Phe derivative aspartame (NutraSweet©). They are labelled to alert those on a Phe-controlled diet.