Nitrogen Fixation & Amino Acid Metabolism Chapter 23
pages 671-690 Outline
• fixing molecular nitrogen from the environment • making amino acids (anabolism)
• the essential amino acids – the ones we can’t make • breaking down amino acids (catabolism)
• throughout the entire course, we’ve come across many molecules that contain nitrogen - amino acids, heme, nucleotides – to name a few
• but, we’ve never discussed the metabolism of nitrogen - only carbon, hydrogen and oxygen
- essentially, the ingredients of sugars • nitrogen is some wicked stuff…
- but that’s only true when it is in some of its forms • in other respects, nitrogen is essential to life
• so, today we will discuss the metabolism of nitrogen
- getting it out of the ________________________ - using it to make amino acids
- converting it into waste and releasing it from the body
• we will not discuss today (or in this course) nucleotide metabolism
General Nitrogen Metabolism
• NITROGEN FIXATION is the process by which ____________________ nitrogen (N2) from the
atmosphere is incorporated into ammonia and then various nitrogen containing _____________ • nitrate ion (NO3-) is another source of environmental nitrogen
- most fertilizers contain this form of nitrogen
• nitrates are reduced to ammonia by the process of ______________________________ - once in ammonia, this nitrogen can again be used to make many organic molecules • DENITRIFICATION converts nitrate and nitrite ions (NO2-) back to nitrogen _______ (N2)
• once made by living cells (either by nitrogen fixation or nitrification) ammonia is converted into
a source of useable nitrogen by ____________
- this form is passed to animals through the _____________________
• animal waste (urine) is composed mostly of urea – another nitrogen-rich molecule (more on that later…)
- urea is converted to ammonia by microbes
• death and decay of animals and plants results in the production of ammonia gas
- denitrifying bacteria reverse the conversion of ammonia to nitrate and then make N2 from that nitrate (as a gas)
Nitrogen Fixation
• all the nitrogen atoms in our body ultimately came from N2 gas in the atmosphere • N2 gas is reduced to NH3 (ammonia) by nitrogen fixation
• there is a ______________ bond between the nitrogens of N2 and they have a combined bond energy of 940 kJ/mol (that’s a lot!)
• nitrogen is very very happy being N2 gas - there is no high transfer potential for anything - it is not reactive
- nothing is gonna happen
• unless something actively goes and makes it happen…
• bacteria are solely responsible for reducing nitrogen gas from the air to ammonia • plants use this ammonia directly to make nitrogen-containing molecules
- in fact, ammonia gas could serve as a potent fertilizer if it didn’t run the high risk of killing the farmers using it…
• the bacteria and some plants form a ___________________ relationship (beans, alfalfa…) - the plant gives the bacteria a safe home
- the bacteria gives the plant ammonia
• many free living bacteria and blue-green algae also fix nitrogen from the air • perhaps, surprisingly – plants and animals cannot fix nitrogen at all
• ~60% of newly fixed nitrogen is done by microbes - lightening and UV fixes another 15%
- we fix the rest via industrial activities…
• industrially, nitrogen fixation requires a catalyst, 500⁰C and 300 atmospheres of pressure (see how hard it is…)
• bacteria do it at room temperature and one atmosphere - amazing…
• the molecular machine responsible for fixing nitrogen in certain bacteria is the _____________ COMPLEX
- it makes ammonia from nitrogen gas - the half-reaction of reduction:
N2 + 8e- + 16ATP + 10H+ → 2NH4+ + 16ADP + 16Pi + H2
• _________ electrons are used to reduce molecular nitrogen to ammonium ion - another two are needed to reduce two protons to H2
• the total reaction is an _________________ electron reduction reaction
• the half reaction of oxidation will not be discussed because different organisms do this process differently
- in the end, the oxidation simply provides the __________________ for this reduction anyway… • the nitrogenase complex contains many redox enzymes (obviously…)
- this includes enzymes with Fe-S clusters and ferredoxin
- electrons flow in one direction – changing hands down the line
approximately ___________ of the ATP generated by green plants from photosynthesis is used for nitrogen fixation
Amino Acid Biosynthesis
• ammonia is highly toxic, so it can’t stick around any living cell for long
- it must be used (converted into something else) rapidly to avoid harming the cell • this is primarily accomplished using the amino acids glutamate and glutamine
- glutamate is made directly from α-ketogluterate (remember…?) - glutamine is made directly from glutamate
• glutamate is made by _______________________ AMINATION • glutamine is made by __________________________
• therefore, glutamate accepts one nitrogen as an amino group - this amino group becomes its α-amino group
• glutamine accepts a second amino group which becomes part of its side chain
• these accepted amino groups can be _________________ to other molecules later on - the transfer of an amino group from donor to acceptor is called a
_____________________________ REACTION
• as usual for any metabolic process, there are a relatively small number of types of reactions used to make amino acids:
- ________________________
- ______________ of one-carbon units (formyl, methyl, etc.)
• in fact, amino acids even share a small number of common precursors
• all of the carbon skeletons of all the amino acids (except one: histidine) come from __________ metabolism
- again, the metabolic hub…
• the citric acid cycle is AMPHIBOLIC
- catabolic: in the sense that it is involved in breaking down glucose
- anabolic: in the sense that it is central to providing precursors for biosynthesis (such as this…) - we touched on this concept already when we wrapped up the citric acid cycle lectures • but, back to actually making amino acids…
• GLUTAMATE __________________________ (GDH) catalyzes the synthesis of glutamate from ammonium ion and α-ketoglutarate
- again, a reductive amination
- a strong reducing agent is needed (energy-requiring process) • glutamate is the amino group ____________ in living cells
- α-ketoglutarate is the amino group _______________ in living cells - this is how nitrogen gets into the system of normal cells
- from ammonia to α-ketoglutarate to glutamate… and then to wherever it is needed • GLUTAMINE ________________________ (GS) catalyzes the formation of glutamine
• these reactions serve to fix inorganic nitrogen (in the form of ammonia: NH3) to form organic (i.e., carbon-containing) nitrogen-containing compounds – such as amino acids
• GDH and GS are responsible for getting the vast majority of nitrogen from ammonia fixed into organic molecules
• OLD MATERIAL ALERT: the KM of GS is much smaller than that of GDH - what does that mean to us…?
- when nitrogen levels are low (often the case for many plants) most of that nitrogen is used by GS to convert already existing glutamate into glutamine
- very little of it is used by GDH to make glutamate from α-ketoglutarate - but this depletes glutamate levels
- glutamate is actually made by a reductive amination reaction where the glutamine side chain is the donor to α-ketoglutarate
• why plants utilize nitrogen in this way when it is limiting is still under debate
• but this is a very good example of how cells keep precursors moving in one direction and/or bias one pathway over another when resources are tight
- having differing KM between enzymes using the same substrate will always give one enzyme an advantage over the other when that substrate becomes limiting
• this is a key and universal concept in biochemistry
• one carbon transfer reactions are almost equally important to amino acid biosynthesis as transamination reactions
• making amino acids of the _________________ family commonly uses 1-C transfers • in addition to being amino acids, serine and glycine often serve as precursors for other
biosynthetic pathways
• making the amino acids of the serine family starts with _______ (our old friend from glycolysis) • first, the hydroxyl group on C2 is _____________________
- then a transamination reaction moves an amino group from glutamate to the substratemaking 3-phosphoserine (and α-ketoglu…)
• the phosphate group is then __________________ to give serine - no 1-C transfers in the making of serine itself
• converting serine to glycine requires a one-carbon transfer
• serine hydroxymethylase catalyzes the transfer of a one carbon unit from serine to an acceptor
- the acceptor is ______________________________ - a derivative of folate (or folic acid) – a vitamin - folic acid is critical for pregnant women…
• the one-carbon unit transferred in this reaction is a ____________________ group - it stays with tetrahydrofolate until this one-carbon carrier gives it to something else - FYI: there are other carriers of one-carbon units (e.g., biotin)
• the conversion of serine to cysteine requires _______________ • in plants and bacteria, serine is first acetylated to form O-acetylserine
- acetyl-CoA is the donor of the acetyl group and the enzyme catalyzing this reaction is SERINE ACETYL-TRANSFERASE
- O-acetylserine is converted into serine with a sulfide group provided by a sulfur donor (3’phospho-5’adenylsulfate)
• animals lack the enzymes required for this pathway, so we make cysteine in different way (methionine is our sulfur donor)
• methionine cannot be made in animals – an ESSENTIAL AMINO ACID - we must take it in through our diet
• therefore our sulfur groups for cysteine biosynthesis come exclusively from our diet
• in the animal cell, methionine reacts with _________ to form a molecule with a high methyl group transfer potential (it is a 1C unit carrier)
- what does this molecule really want to donate….?
• once this molecule does give up its methyl group (to anything…) it becomes S-adenosyl-homocysteine (we’re getting close…)
- hydrolysis of this molecule yields homocysteine
• serine and homocysteine then come together and, after one more step, make cysteine (and α-ketobutyrate)
- this pathway to make cysteine is exclusive to ______________________ The Essential Amino Acids
• all 20 amino acids are needed to make the proteins required for life - using our analogy from the second week of this semester…
… imagine trying to communicate in English if one letter went missing from the alphabet • E. coli can make all 20 amino acids ‘from scratch’ using individual atoms and groups from other
molecules - we can not…
• this poses a bit of a paradox
- we need all twenty amino acids to make proteins, but we can’t make all twenty - the amino acids we cannot make, we MUST obtain from our diet
- we are, quite literally, fully dependent on the food we eat for survival - it is essential that we get these amino acids from our food
- therefore, they are called the ESSENTIAL AMINO ACIDS • some of the ‘essential’ amino acids we can make
- but not in quantities sufficient to support protein synthesis • this is especially true in growing children
• there is no storage form for excess amino acids - nothing like glycogen or adipose tissue
• therefore, your existing proteins are the only source of amino acids… - life will digest itself if necessary…
Breaking Down Amino Acids
• the first step of any real importance when breaking down amino acids is __________________ - getting that amino group off!
• that amino group always goes to α-ketoglutarate (the universal acceptor of nitrogen in our bodies) making glutamate
• what’s left behind of the original amino acid is just carbon and is called the CARBON SKELETON • we will discuss what happens to the carbon skeleton and the amino group separately
- but remember that, initially, these came from the same single amino acid • the carbon skeletons of amino acids can go down one of two roads :
oxaloacetate
- oxaloacetate is the first precursor for making glucose by gluconeogenesis, so these amino acid skeletons may eventually become glucose (hence, the name) - a ____________________ amino acid has its carbon skeleton become acetyl-CoA or
acetoacetyl-CoA
- these will eventually give rise to ketone bodies (hence…)
• amino acid skeletons can also become many different intermediates that we’ve seen before (i.e., α-ketoglutarate, fumerate, succinyl-CoA and the others mentioned above)
• essentially, metabolically speaking, the glucogenic amino acids can be used to make
______________ (if necessary); the ketogenic can only enter the ____________________ and be used for energy (ATP)
• excess nitrogen no longer needed by the body is excreted as ammonia, urea, and uric acid (or a combination of these)
• aquatic animals (e.g., fish) excrete straight ________________ because it is rapidly diluted in their watery environment
• most terrestrial animals (including us) excrete nitrogen primarily as urea
- urea is less toxic and water-_______________
• however, this means that terrestrial animals must also carry ____________ around with them in order to have something to put the urea in
• can anyone think of an animal that cannot afford to carry water around for this purpose…?
- _____________ excrete nitrogen as uric acid – this requires no water and keeps ________________________
The Urea Cycle
• this cycle will allow us to gather our excess __________________ and use it to make urea – and then eventually to be excreted in urine
• the nitrogen entering this cycle comes from ____________________ - ammonia is released by glutamate dehydrogenase
- but glutamate (formerly α-ketoglutarate) received this nitrogen from anywhere and everywhere
• part of this cycle occurs in the mitochondria, while the rest occurs in the _______________ • mitochondrial glutaminase can provide free ammonia for the urea cycle as well
• let’s make some urea!
• first, a condensation reaction occurs between ammonia and CO2 making __________________ PHOSPHATE
- this reaction requires two ATPs
- getting rid of nitrogen is a _____________________ for the cell (worth some ATP) • _____________________ phosphate then reacts with ornithine to form citrulline
- this is the first step of the urea cycle
• at this point citrulline is transported to the _________________ • aspartate then reacts with citrulline to form argininosuccinate
- this step also burns an ATP
• argininosuccinate is then split to form arginine and ____________________ (step 3) • finally, arginine is hydrolyzed to yield urea and regenerate ornithine
- this ornithine is transported back to the mitochondria so that the cycle can start again • fumerate is a link between the __________________________ and the urea cycle
• if one cycle is going strong and the other is needed, one cycle can feed the other intermediates to compensate
• after a high-__________________ meal the body is likely to have too much nitrogen - the citric acid cycle will sped up as the urea cycle ‘donates’ some of its fumerate while also
getting rid of that excess nitro…
• in humans (and other higher eukaryotes), the urea cycle is restricted to the _____________ • but – in my mind – it’s the crosstalk that is fascinating… the complexity!
Summary
• NITROGEN FIXATION is the process by which N2 from the air is made into ammonia - bacteria are responsible for making this ammonia
• the molecular machine responsible for fixing nitrogen in certain bacteria is the
__________________________________ COMPLEX
• glutamate is the amino group _______________ in living cells • α-ketoglutarate is the amino group ______________ in living cells
• transamination and one-carbon unit transfer reactions are most commonly used when synthesizing amino acids
• the amino acids we cannot make, we MUST obtain from our diet - called the ESSENTIAL AMINO ACIDS
• the first step of any real importance when breaking down amino acids is __________________ - getting that amino group off!
• the carbon skeletons of amino acids can go down one of two roads :
- GLUCOGENIC become ____________
- KETOGENIC become ____________________ or acetoacetyl-CoA (______________________) • the urea cycle allows us to gather excess __________________________ and use it to make
