Who doesn’t love the smell of maple syrup? Doctors who smell it coming from diapers! Don’t know what i’m talking about – feeling sorta alone? It’s a bad pun about sotolone! Sotolone is a rare molassesy smelling side product of protein letter (amino acid) breakdown. In particular, sotolone can be made during the breaking down of  branched chain amino acids (BCAA) including ISOLEUCINE (Iso, Ile), and it builds up when people have mutations in the branched-chain a-ketoacid dehydrogenase complex (BCKD complex). Those mutations prevent BCAAs from being fully broken down into their normal products. And it’s a telltale sign of Maple Syrup Urine Disease (MSUD) – aka branched-chain aminoaciduria.

video added 12/5/21

It’s Day 5 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 Isoleucine!

As the similar names suggest, the amino acid Isoleucine (Ile, I) is a lot like yesterday’s Leucine (Leu, L) which was a lot like the previous day’s valine (Val, V) – starting with the composition of their side chains (R groups) (the part that makes each amino acid unique). These 3 amino acids constitute the Branched Chain Amino Acids (BCAAs) which have branching hydrocarbon chains. As that term suggests, these chains are made up of carbon and hydrogen atoms. Atoms join together to form molecules by sharing pairs of electrons, which are negatively-charged subatomic particles. Hydrogen & carbon share electrons pretty fairly, which makes them “nonpolar” (meaning they have even charge distribution) and thus hydrophobic – they’re excluded by the surrounding water, forcing them to seek refuge in the protein’s interior (so don’t expect to find them on the surface of a protein).

Valine’s R group is an “isopropyl” group (propyl meaning 3) – 3 C’s sticking out like a V. Stick a methylene (CH₂) in there to push the V further away so it’s more like a Y and you get leucine. Move the branch point in 1 and you get isoleucine. We call this R group a “sec-butyl” group, a 4 carbon (C)(“butyl”) chain attached 2 backbone at its SECond C. The bulkiness close to the backbone limits its flexibility, so like valine, it doesn’t like to form helices. 

The similarities of these 3 amino acids don’t stop with shape and water-unhappiness. They are all “essential” amino acids, meaning that your body can’t make it so you need to get it from your food (you still need the “nonessential ones, you just don’t need to get them “pre-made” in your food! 

They also share breakdown (catabolism) machinery. So if something goes wrong with one of these shared machines, the breakdown of all 3 is impacted, and this is the case with Maple Syrup Urine Disease (MSUD) – aka branched-chain aminoaciduria. MSUD is an “inborn error of metabolism” (genetic problem with processing biochemicals) which was first described in 1950s. It doesn’t affect the processing of *all* biochemicals – instead it affects the breakdown of branched-chain amino acids (BCAAs) – valine and leucine, which we’ve already seen, and isoleucine, which we’re looking at today.

MSUD only affects those 3 (but it does affect all 3 of them) because it is caused by defects in the branched-chain a-ketoacid dehydrogenase complex (BCKD complex), which they all need for complete breakdown. This “factory problem” results in elevations of BCAAs in plasma and their upstream intermediary products, α-ketoacids, in urine, as well as production of a “sterically-flipped” version of isleucine called alloisoleucine, where, depending on your point of view, the branch of the chain sticks “out” or “back.” 

A “side product” called sortolone also builds up in the urine and gives the distinctive maple syrup smell, which is often how doctors tell. But that’s far from the worse sign or symptom. Signs and symptoms vary depending on what mutations are present and how “bad” they are – in the worst case, the “classical” case, there’s practically no functional BCKDH made, so the metabolic traffic jam becomes detectable soon after birth, with signs involving developmental delay, problems feeding, and (how could you not have) “failure to thrive.” In less severe cases, some BCKDH is made, just not enough to keep up in times of high demand, so signs often don’t get recognized until later in life, and usually during metabolically stressful times.

Amino acids can get into the brain via specialized amino acid transporters. BCAAs enter through a transporter called LAT1. But multiple amino acids (large, neutral ones) have to share this “single door.” There are lots of copies of this “door” but there are LOTS more amino acids trying to get in. Just like a crowd on Black Friday, there can be quite a traffic jam to get in, with the more “aggressive” molecules “jumping the line”

Leucine is really good at forcing its way in – it’s not like there’s really a “line” of amino acids waiting to get in, its just that the channels have a higher affinity for it (they like each other more so if a leucine collides with it, it’s more likely to get let in). The more leucine there is floating around, the more likely it is to collide and the more likely it is to collide, the more likely it is to be let in. And consequently the less likely other amino acids are to get in. 

This can be a problem because some of those amino acids that are getting left out are REALLY important for your brain. Especially glutamate, which acts as a neurotransmitter (brain signaling molecule) and tryptophan, which is a precursor to dopamine & serotonin. And you also need those other amino acids to make the proteins you want. So, by outcompeting these other amino acids, the BCAAs mess up brain signaling. And they also screw with water balance by depleting ATP which is needed to maintain ion balance via the sodium-potassium pump. So, with BCAA build-up, more water enters the brain causing swelling (cerebral edema). 

MSUD affects 1 in 185,000 people worldwide and untreated, “classic” MSUD is fatal. Treatment depends on the severity – the standard therapy is a diet low in BCAAs and, in severe cases, liver transplant. It’s autosomal recessive meaning that you have to get one faulty copy of the gene from each parent for symptoms to show. But the mutations don’t have to be the same, they just can’t compensate for one another. And, as we’ll see, there are multiple genes involved, so there are a lot of ways things can go wrong (it’s genetically heterogeneous), with over 60 mutations identified.

Ready to dive in?

The 1st step to breaking down an amino acid is removing the nitrogen – if you look at the structure of sugars and fats, you won’t see nitrogen there. So if you want to make those things from it you’ll have to ditch the N. And, for the BCAAs, this is accomplished by Branched-Chain AminoTransferase (BCAT), which works fine in MSUD patients, whose problem lies further downstream. 

You don’t just want to chop off the amine group to make ammonia (NH₄⁺) because that’s toxic to your cells. Instead, if you want to get rid of that nitrogen you’ll have to pass it off carefully from molecule to molecule until you can ditch it in your pee as urea.

The first pass-off comes from BCAT transferring the amine from a BCAA like leucine to α-ketoglutarate. α-ketoglutarate is one of the products of the citric acid cycle (TCA) which is part of the sugar breaking down process. It’s basically the amino acid glutamate but with a carbonyl (C=O) instead of an amino group. So if you swap those out you get glutamate. The BCAA provides the amine for the swap and BCAT helps with the transfer. The glutamate can then go off and get rid of the nitrogen through the urea cycle (or it can be used as glutamate). 

And the leftovers, the deaminated BCAA, having lost its amino, is now no longer an amino acid – instead it’s a branched-chain α-keto acid (BCKA). In the case of isoleucine, you get α-keto—β-methylvaleric acid (KMV). The “keto” refers to “ketone” which is a carbonyl (C=O) hooked up to carbons on either side (so -C-(C=O)-C-). It gets to keep the “acid” part of its name because you haven’t touched the carboxylic acid group. 

These α-keto acids can then, IN HEALTHY PEOPLE, go through further processing with the help of the Branched-Chain α-keto acid dehydrogenase (BCKDH) complex, which “oxidatively decarboxylates” it – yowza, that’s a mouthful. It basically means that it removes electrons (oxidation) from the a-keto acid and it removes the carboxylic acid group (decarboxylation). The “stolen” electrons get passed first to BCKDH and then to an NAD⁺ carrier which can go off to cash them in for ATP (cellular energy money). 

Depending on which BCAA went in, you’ll get left with different products which can have different fates. In the case of isoleucine, you’re left with α-methylbutyrl-CoA, which can get further processed to give you acetyl-CoA and succinyl-CoA. acetyl-CoA can be used to make ketone bodies and lipids, so we call it ketogenic. And the succinyl-CoA can get used to make glucose (blood sugar), so we call it glucogenic. So isoleucine is both ketogenic and glucogenic. (leucine is strictly ketogenic, whereas valine is strictly glucogenic, but isoleucine’s both). more on this ketogenic vs. glucogenic business here: http://bit.ly/luckyleucine 

So, Branched-Chain α-keto acid dehydrogenase (BCKDH) complex… As the mouthful of a name suggests, the process it carries out is multi-step & requires the help of multiple protein enzymes (reaction mediators/speed-uppers), any one of which can have mutations that can cause traffic jams (or traffic syrups?) that lead to the buildup of BCAAs & α-ketoacids (remember they can still do that first BCAT step).

The BCKD complex has multiple copies of several enzymes (it has to do several things so it’s more like a factory assembly line than an individual robot), which are loosely held together in the inner mitochondrial membrane (mitochondria are like your cell’s power plants – they are where TCA & oxidative phosphorylation take place & produce ATP).

BCKDH has 3 “catalytic subunits.” Catalysis refers to the speeding up of reactions by something that doesn’t get used up in the process (so it facilitates a reaction, maybe by holding the reactants together and in the right orientation to react) but at the end of the reaction it can do it all again. And again. And again. The 3 catalytic subunits of BCKDH are creatively named E1, E2, & E3 (not everything can have fun names like ammonia (which I learned the other day was “derived from the deity Jupiter Ammon, near whose temple in Libya this substance was first prepared by the distillation of camel dung” https://t.co/t4BibsYv1v?amp=1 )

So, “E1,” “E2,” & “E3” – lacks creativity… But these short names are easier to say than their “full names,” which for the case of E1 is branched-chain α-keto acid decarboxylase. E1 is itself made up of multiple parts (this complex is indeed complex…) It is a heterotetramer, which means it’s made up of 4 (tetra) subunits, some of which are different (hetero) – each “E1” has 2 α subunits and 2 β subunits (coded for by the BCKDHA/B genes). So, instead of having a single folded up polypeptide chain, they have 4 chains. These chains are held together to give the complex its so-called “quaternary (4°) structure” and they work together to decarboxylate the BCAAs using the small non-protein helper molecule thiamine as a “cofactor”. 

Decarboxylation takes away a carboxylate (-(C=O)-O-) group, leaving you with an acyl group (what an ass, right? wrong – it’s all part of the process!). That acyl group is then transferred to a lipoamide cofactor bound to E2 (basically a little fatty thing hooked onto the N of one of E2’s lysines), which will pass it off to CoA to form acyl-CoA in a molecular relay of α-ketoacid -> E1 -> E2 -> CoA

E2 is dihydrolipoyl transacylase. And if you thought E1 was “chain-y” with it’s 4 protein chains, wait till you meet this guy – it has 24! It provides a strong structural core helping hold everything together. But it’s not just a scaffold. It’s also catalytic – it passes the acyl group it’s been given to the next in line – CoA, to form acyl-CoA which can go off on it’s way.

Done right? Why do you need E3? Well, remember how I said enzymes can’t be “used up”? Well, E2 now has its lipoamide in a “reduced” state (it has “extra” electrons), so it needs to be converted back, and this is the job of E3, dihydrolipoyl dehydrogenase (DHD). DHD, using a bound FAD cofactor and NAD⁺ as an electron acceptor, takes those electrons, “resetting the system.” 

E3 is “only” a dimer – it has 2 protein chains – and these chains are the same, so we call it a homodimer. But it’s got multiple uses. Unlike E1 & E2 which work exclusively for BCKD, E3 is shared by other α-keto acid dehydrogenase complexes (the pyruvate dehydrogenase complex & the α-ketoglutarate dehydrogenase complex). So mutations in E3 can cause problems with more than “just” BCAA breakdown. 

What about those other 2 guys? BCKDK & PPM1K are regulatory subunits. BCKDK is a kinase that inactivates BCKD by phosphorylating E1α. And PPM1K (protein phosphatase, Mg²⁺/Mn²⁺ dependent 1K) is a phosphatase that removes that phosphatase to reactivate it. 

The whole complex has that big E2 core surrounded by 12 E1 subunits & 6 E3 subunits, so the whole shebang is a whopping 4 megadaltons (MDa). Which is really really big (for comparison, I study a medium-sized protein that’s ~100 kDa, and a megadalton is a thousand kilodaltons, so this thing is ~400 times bigger, but that’s mainly because it has so many different proteins in it.

Since there are a lot of genes involved there are a lot of ways things can go wrong – over 60 mutations have been identified and MSUD can be characterized by where the mutations are.

  • Type IA: mutations in E1α 
  • Type IΒ: mutations in E1β
  • Type II: mutations in E2
  • Type III: mutations in E3 

As for the sweet-smelling sotolone (sotolon). I tried to find more information about how it forms and accumulates but my searches mainly turn up “is spontaneously produced.” So it seems like it’s not a normal intermediate of breakdown and it doesn’t have an enzyme dedicated to making it specifically but when you have a buildup of a bunch of BCAA’s, they and/or their breakdown intermediates can “get bored” and interact with other molecules and rearrange themselves. Okay, not really like that, but all molecules can undergo rare reactions, but you won’t detect them unless you have lots and lots of potentials for those reactions to occur. It’s kinda like how you do drug trials on really large numbers of people so that you can detect the really rare side effects. So maybe that’s the sort of situation you have here, but on the really small scale. Additionally, enzymes can get tricked into acting on some of these side-products, etc. The main point is that you need the accumulation of the precursors in order to see (or smell…) them. If you want to get super in-the-weeds geeky, the paper that discovered it as the smelly thing in the pee (Podebrad et. al 1999) offers a couple possible synthetic routes https://link.springer.com/article/10.1023/A:1005433516026 

Ile-arned a lot about isoleucine for this post. And here are some final notes…

Ehrlich discovered isoleucine in 1903 and through a lot of careful chemistry correctly characterized its structural composition a few years later. https://doi.org/10.1021/cr60033a001 

how does it measure up?

coded for by: AUU, AUC, AUA
empirical formula: C6H13NO2
molar mass: 131.175 g·mol−1

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0

more resources:

stereochemistry https://biosci.mcdb.ucsb.edu/biochemistry/tw-amn/isoleucine_stereo_diastereoisomers.htm 

medical biochemistry page: https://themedicalbiochemistrypage.org/maple-syrup-urine-disease-msud/ 

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