I’ve always been more interested in the super close and super small than the super far and super big. So, while some may dream of space shuttles I dream of malate-ASPARTATE shuttles! Aspartic acid or aspartate? Depends on the protonation state – either way, be sure to get it because it’s your ticket from the mitochondrial matrix to the cytosol! Sound boring? It’s actual essential for energy making! And it’s really cool, so hopefully learning about it will energize you!
It’s Day 16 of #20DaysOfAminoAcids – the bumbling biochemist’s version of an advent calendar. Amino acids are the building blocks of proteins. There are 20 (common) genetically-specified ones, each with a generic backbone with to allow for linking up through peptide bonds to form chains (polypeptides) that fold up into functional proteins, as well as unique side chains (aka “R groups” that stick off like charms from a charm bracelet). Each day I’m going to bring you the story of one of these “charms” – what we know about it and how we know about it, where it comes from, where it goes, and outstanding questions nobody knows. ⠀
More on amino acids in general here http://bit.ly/aminoacidstoproteins but the basic overview is: amino acids have generic “amino” (NH₃⁺/NH₂) & “carboxyl” (COOH/COO⁻) groups that let them link up together through peptide bonds (N links to C, H₂O lost, and the remaining “residual” parts are called residues). The reason for the “2 options” in parentheses is that these groups’ protonation state (how many protons (H⁺ ) they have) depends on the pH (which is a measure of how many free H⁺ are around to take).⠀
Those generic parts are attached to a central “alpha carbon” (Ca), which is also attached to one of 20 unique side chains (“R groups”) which have different properties (big, small, hydrophilic (water-loving), hydrophobic (water-avoided), etc.) & proteins have different combos of them, so the proteins have different properties. And we can get a better appreciation and understanding of proteins if we look at those letters. So, today let’s look at Aspartic acid/aspartate (Asp, D)
First, let’s clear up that naming thing. You’ll see this amino acid listed as aspartate or aspartic acid. A lot of times the terms are used pretty willy-nilly, but the naming difference has to do with whether or not its side chain is “protonated.” All free amino acids have at least one carboxyl group in the generic part, attached to the central carbon, Cα. It can be in the form of a carboxylic acid group – a carbonyl (C=O) attached to an -OH. Or, if it gives up that H⁺, it now has a “carboxylate group” (C=O)-O⁻. When something gives up an H⁺ (proton) we say it acts as an acid, and when something takes a proton, we say it acts as a base. And this proton giving and taking is reversible – two sides of the same coin – so “carboxylate” is the “conjugate base” form of “carboxylic acid”.
When amino acids link up through peptide bonds to form proteins, they lose the (-OH/O) part, but 2 of the amino acids, Asp & Glu, still have a carboxyl group even when they link because they have an “extra” one in their side chain. And, just like the free amino acids can be in the carboxylic acid or carboxylate forms, so can the carboxyl groups in these side chains. So they each can have 2 forms with different names – aspartic acid and glutamic acid are protonated – so they have the ability to act as an acid (donate a proton) whereas aspartate and glutamate have already acted as an acid, so now they’re the “conjugate base” forms. These forms are able to take back a proton if there are plenty around (low pH) but if the proton supply is scarce (high pH) they’ll stay unprotonated.
In their unprotonated state, they’re negatively-charged. This is because atoms join through strong covalent bonds by sharing electrons (negatively-charged subatomic particles that whizz around dense central atomic nuclei containing positively-charged protons & neutral neutrons) and the H leaves its electron behind when it leaves as a proton, so you’re left with an imbalance of protons and electrons.
The carboxylic acid in Asp’s side chain is separated from the backbone by a methylene (-CH₂) linker & has a pKa of 3.65. This means that at a pH of 3.65, 1/2 of Asp will be protonated & half won’t be. When you get below 3.65, the protonated form (aspartic acid) becomes more common. But when you go above 3.65, the deprotonated, negatively-charged, form (aspartate) dominates. And the pH inside your cells is around 7.4-ish, which is well above 3.65, so this aspartate form dominates in the free-floating stuff, and this is how it’s “billed” in the credits for today’s big show, the MALATE-ASPARTATE SHUTTLE!
You need a way to convert the energy you can get from burning food fuel to a form you can spend when you’re ready, at biochemical “point of sale” transactions. And nature’s decided Adenosine Triphosphate (ATP) is great for this because it has 3 phosphate groups (phosphorus surrounding by 4 oxygens) stuck together (and stuck to the sugar-nucleobase combo adenosine). Each of those phosphates is negatively charged so, since opposites charges repel, they want to get apart from each other. Therefore, like clamping together a stiff spring, it takes energy to keep them bonded together (we call this chemical potential energy)- if you let one go (split ATP into ADP + Pi (inorganic phosphate)) you release energy, and you can use that energy to do things like build proteins and stuff.
So our cells use ATP kinda like arcade tokens, but you have to buy the tokens. And the “money” can be coming from all different fuel sources (sugars, fats, proteins). Analogous to using different currencies and denominations to purchase arcade tokens, you need a way to convert the energy from all those sources into to the universal ATP currency. Enter the electron. All molecules (except protons) have them, so this really is the universal universal currency of the inter-atomic world.
Molecules can pass electrons off to one another. “Passing of electrons” is called OXIDATION. When something gives up electrons, we say it gets oxidized and when something accepts electrons, we say it’s been reduced – they always go hand in hand so we call such passings REDOX reactions. You can remember which is which with the acronym OIL RIG: Oxidation Is Loss (of electrons); Reduction is Gain (of electrons). Much more on redox here: http://bit.ly/2Yiya50
Similarly to how ATP is a “universal energy source”, NADH is a sort of “universal electron carrier” Nicotinamide Adenine Dinucleotide (NAD⁺) and it’s cousin NADP⁺ (just add phosphate) can accept electrons (get reduced) to become NADH and NADPH. It might look like the big difference between NAD⁺ and NADH is a hydrogen (H), but the real difference is “unspoken” – it’s the electrons! When NAD⁺ picks up that H, it picks it up as something called a HYDRIDE, which is an H with 2 electrons instead of its usual 1. Hydrogen atoms only have 1 proton & 1 electron & they often leave that electron behind when they go, so all they’re left with is a H⁺, so we often call H⁺ a proton but when H’s have 2 electrons we call it a hydride
NADH makes a great carrier because it wants electrons, but not desperately, so it’s willing to give and take. Some molecules want electrons more than others – and if something which doesn’t want or only kinda wants an electron (has a lower reduction potential) gives an electron to something that’s happier with it than it is (has a higher reduction potential) you get a net gain in happiness. And when you make molecules happier, they release energy (I like to think of it as them not having to fidget as much to get comfy so they can relax). So you can pass electrons from things with low reduction potential (like NADH) to things with high reduction potential (like O₂), and release little bits of energy as you do, which you can put to use – including to make ATP!
Electron-to-ATP conversion occurs in a process called OXIDATIVE PHOSPHORYLATION, which takes place in a secret club house in your cells called the mitochondrium (your cells actually have lots of mitochondria because of their important roles as cellular powerhouses). Unlike some other organelles (membrane-bound compartments inside your cells) like the endoplasmic reticulum (involved in protein processing), mitochondria have double membranes because they come from an ancient ancient ancient cell swallowing a bacteria and then adapting that bacteria to suit its needs, specializing in energy production through oxidative phosphorylation (oxphos).
OXIDATIVE PHOSPHORYLATION consists of an ELECTRON TRANSPORT CHAIN (ETC) and CHEMIOSMOSIS. The electron transport chain (ETC) occurs at the inner membrane (between the inner matrix & intermembrane space). Starting with NADH (or FADH), the original electron carrier transfers its electrons to another molecule that wants them more which passes them to another molecule that wants them even more . . . until they reach oxygen which wants them the most. Each of those pass-offs releases a little energy and that energy is used to pump protons out of the inner room of the mitochondria (mitochondrial matrix) into the outer room of the mitochondria (intermembrane space), creating a proton gradient where there are more protons in the intermembrane space than there are in the matrix.
Only one way back in is provided – through the ATP-making factory, ATP synthase, or as I like to call it, the ATP ATM. ATP synthase is seriously one of the most incredible machines ever made, able to harness the power of flowing protons similarly to how a hydrolytic dam harnesses the power of flowing water, in order to turn a molecular crank and turn ADP + Pi into ATP. It’s way more efficient than any man-made machine – and way way tinier. For each NADH that goes in, ~10 protons get pumped out, and it takes 4 protons coming in for 1 ATP to be made. 10/4= 2.5, so you get ~2 and a half ATP per NADH.
Pretty cool, eh? But you have to get the NADH in there (into the mitochondrial matrix) and spoiler alert – you can’t… At least not directly – but you can pass along the electrons its holding onto for you (which is the main thing you care about anyway). And then you can use those electrons to reduce more NAD⁺ inside the matrix so you end up with NADH in there without it ever crossing the inner mitochondrial matrix – sneaky, eh?
Why can’t you just take the NADH in from where it’s made in the early breakdown steps of food? For example, in the early sugar breakdown process glycolysis, which takes place in the cytoplasm, you get 2 molecules of NADH (from NAD⁺). But you can’t ship them in because it’s purposefully hard to get into the mitochondrial matrix. You can’t just let any ole molecule in, or else what’s the point of having a compartment anyway, right? So the inner mitochondrial membrane (IMM) is selective about what it lets in (it’s much more selective than the outer mitochondrial membrane so molecules that can pass through generic pores in the outer mitochondrial membrane (OMM) need specialized channels to get through the inner one). But the membrane only has control at the import/export stage – it can choose what to let through but can’t police what the molecules do once they’re in or out…
So your cells find a way to to get those electrons in without taking in NADH itself. Malate dehydrogenase in the cytoplasm transfers electrons generated in the cytoplasm (such as by glycolysis) from NADH to oxaloacetate. Going back to OIL RIG, we can say NADH got oxidized (lost electrons to become NAD⁺) and oxaloacetate got reduced (gained electrons to become malate. This has the effect of transferring 2 electrons (and a H⁺) from NADH (which can’t cross the IMM) to something that can (malate).
So malate enters, and it does so through an antiporter – a type of membrane passageway that does a swap where one thing comes in in exchange for another going out. In this case, malate comes in and alpha-ketoglutarate leaves. So you’ve gotten malate into the mitochondrial matrix – the next goal is to get the electrons it’s transporting to the ATP ATM. And that factory wants those electrons to come from NADH, so now you have to reverse what you had to do to get in. With the help of malate dehydrogenase (the mitochondrial type this time) you take those electrons back out of malate and plop them back onto NAD⁺ (a different copy of it of course, but there’s tons floating around) to give you NADH. This NAD⁺ reduction generates oxaloacetate again.
The NADH is now in the proper location for getting to the ATP factory, so it goes off and gets converted into ATP via oxidative phosphorylation. And now you’re left with the “leftovers” as oxaloacetate. It’s not useful in the matrix, but it can be reused out in the cytosol to transfer electrons again. So you want to ship it out there. But you have a similar problem to the one you had in the beginning. Oxaloacetate can’t get through, so you have to convert it to something that can get through.
This time, instead of converting it to malate, you convert it to aspartate, getting the amino group from the amino acid glutamate. When you take away glutamate’s amino group you get alpha-ketoglutarate (this is the molecule we can exchange malate for in the first swap). This time, in the aspartate swap, we use a different antiporter – the glutamate-aspartate antiporter. It brings in glutamate when you send out aspartate (which is good because we need to replenish the glutamate!)
But the cytosol might not want aspartate – and if it does use aspartate for something else, you’re not regenerating the oxaloacetate. So, if you want to keep the cycle going strong you can use cytosolic aspartate aminotransferase to deaminate aspartate back to oxaloacetate, plopping the amino group off onto alpha-ketoglutarate to replenish your glutamate. So basically this malate-aspartate shuttle allows you to interconvert over and over again between the same molecules, with only electrons really getting used up (because they get taken out of the cycle to be used for oxphos). This malate-aspartate shuttle is the main way of moving electrons from NADH into the mitochondria in the liver, heart, & kidneys. Some tissues use other ways.
Awesome right? And that is just one of many ways aspartate’s super useful…
Even though *free* Asp is usually in the deprotonated, aspartate, form, when it’s in proteins it can get greatly influenced by what’s nearby and can thus be used to give & take protons in the “active site” of enzymes (proteins (or protein/RNA complexes, or just RNA) that speed up chemical reactions). It can give & take H⁺ from reactants to “activate” them and it can stabilize reaction intermediates so they can get past energy barriers.
It also plays central role in nitrogen metabolism, by connecting the energy-releasing CITRIC ACID CYCLE (aka “Krebs cycle” aka “tricarboxylic acid cycle” (TCA)) to the excess-nitrogenous-waste-removing UREA CYCLE (this connection’s sometimes called “Krebs bicycle”). Breakdown (catabolism) of amino acids produces ammonia (NH₃). Ammonia’s toxic, so our bodies convert it to the less toxic UREA. TCA takes place in the mitochondria & oxaloacetate produced by TCA can can get transaminated (C=O replaced w/amino group) to form Asp, which shuttles out of the mitochondria & gets converted into argininosuccinate, which can enter the urea cycle. This is called the aspartate-argininosuccinate shunt.
Asp is a precursor to several other amino acids – methionine, threonine, isoleucine, & lysine. It also helps make DNA/RNA pieces (it donates a nitrogen in the making the purine base precursor, inosine) & it’s GLUCOGENIC (can be made into glucose (sugar)).
Aspartate is NONESSENTIAL, which doesn’t mean we don’t need it, it just means our bodies can make it – and they can do it either by transaminating oxaloacetate (aspartate transaminase, AST can take the amino group from glutamate & stick it on oxaloacetate) or by delaminating asparagine (with the help of asparaginase, which releases the -NH₂ as ammonia).
Speaking of making it from asparagine… Asp was first discovered from the hydrolysis of asparagine (by Plisson in 1827). That asparagine was actually the first amino acid to be isolated (in 1806 by Vauquelin and Robiquet from asparagus juice, hence the name). But they didn’t know asparagine was a protein letter at that time, and Plisson didn’t know that aspartate was a protein letter either. In fact, when naming aspartic acid, Plisson chose “aspartic” instead of “asparagic” to emphasize that at that point they didn’t know if it really was a natural thing so they didn’t want people to confuse aspartate for being a natural product of asparagine (which we now know it is). In 1866, Ritthausen was the first to isolate aspartate from the products of protein hydrolysis (the proteins he used were conglutin & legumin) and then Kreusler found it in animal proteins too – in casein (these stories can never escape casein, can they?) & egg proteins. https://doi.org/10.1021/cr60033a001
Speaking of naming, aspartate and aspartic acid are the “same” give or take a proton – but aspartame is more than just a difference in name, it’s a whole different ball game! Aspartame is an artificial sweetener. We already saw it once in our amino acid advent, when we where looking at phenylalanine. Aspartame consists of aspartate & phenylalanine joined together and people with the genetic disease phenylketonuria (PKU) can’t break down phenylalanine, so products with aspartate have to put a “heads up” on their products telling people that there’s phenylalanine in there (or at least there will be when your body digests it). On a molecule by molecule basis it’s way sweeter than sugar so, even though it still has some calories, you have to use such little amounts of it that they’re often considered “calorie-free” http://bit.ly/pkustoryandscience
How does it measure up?
systematic name: 2-Aminobutanedioic acid
coded for by: GAU, GAC
chemical formula: C4H7NO4
molar mass: 133.103 g·mol−1