Three cheers for THREONINE! Once scientists identified the first amino acids, the race was on – Pokemon Go amino acid style – gotta find them all! And the last to be found and characterized was Threonine (Thr, T). It’s a pretty thrilling tale that will hopefully never get stale! It was last to be discovered but I couldn’t wait to tell you about it, especially when I found out that grad students were literally essential to figuring that out threonine is essential to the human diet…  

blog form (refreshed from last December):

It’s Day 6 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 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 threonine!⠀

Threonine (or as its friends call it Thr, or just T) has three “groups” in its side chain, and one of them has an O. But that’s not why it got that name – the name has a less mnemonically-satisfying origin – structural relationship to the 4-carbon carbohydrate “threnodic acid,” which gets its name from coming from the sugar threose, which gets its name from scrambling up the letters of a sterically-scrambled (atoms stick off other atoms in different directions) version of this sugar, erythrose, which gets its name because it turns red in basic solutions. If you’re interested in etymologies, I found this awesome website, where I learned this

Speaking of origins, I want to tell you about how threonine was discovered, but first I want to tell you all about threonine biochemistry-wise, and how it provides opportunities for proteins to get “modified” after they’ve been made. I know, I know, I’ve been rambling on and on about how important the primary sequence (order of amino acids) is to proteins because their unique parts affect how they fold up. But now I’m telling you that parts of the unique parts can be changed?! Don’t worry – I haven’t been lying to you about the primary importance of primary structure – only specific amino acids can be altered, and threonine’s one of the them, so the amino acid sequence matters even here!

So what’s so special about threonine? Threonine looks like valine which, as you might remember, has that “V” of a side chain where each “point” on the V is a carbon/hydrogen group – so 1 methylene (CH₂) branching off into 2 methyl (CH₃) groups. Threonine also has a V, but there’s a BIG difference. Instead of 2 methyls, one of the branches is a hydroxyl (-OH) group. This makes Thr an “alcohol.” When you hear the word “alcohol” you probably think of wine, or beer, etc. but that’s just 1 kind of alcohol (ethanol) – the term “alcohol” just refers to something that has one or more hydroxyl (-OH)) groups.

Why does this matter? Molecules are formed by atoms linking up through strong covalent bonds, which are formed by atoms sharing electrons. Electrons are negatively-charged subatomic particles that whizz around each atoms’ dense central core called the nucleus, which contains positively-charged protons (and neutral neutrons). Some atomic nuclei keep a “tighter leash” on their electrons (including the ones which they share), which can uneven the charge balance, in a phenomenon we call POLARITY. Since opposite charges attract, this can lead to partially charged regions being attracted to other partly or fully charged things with the opposite charge. 

A great example of a polar molecule is water. Oxygen (O) is a major electron hog (it is highly electronegative). So when it hooks up to hydrogens through shared pairs of electrons it pulls the electrons towards it, leaving the H’s with less. So the O becomes partly – (δ⁻) & the H’s partly + (δ⁺).  Those oppositely-charged parts of different water molecules attract one another, leading water to be “self-sticky” and form large, interconnected networks. They’ll only let other molecules hang out with them if those other molecules have something as good as or better to offer. 

If we look back at valine, we see that it has a pure “hydrocarbon” side chain – it only contains Carbon (C) & hydrogen (H) atoms. C & H share electrons pretty fairly, so in valine’s methyl groups, since there’s an even # of protons & electrons (which have equal but opposite charges) and those electrons are evenly spread out, there is “no charge” anywhere. Therefore, we call valine (and other hydrocarbon chain having amino acids like leucine & isoleucine) NONPOLAR. They have “nothing to offer” the highly polar water around them, so they tend to get excluded and seek refuge deep in the protein’s central core.

BUT in threonine, one of the methyl groups is swapped out for an -OH group. And, as we saw with water, O pulls the shared electrons away from the H, making the O partly – (δ⁻) & the H partly + (δ⁺). Therefore, threonine is classified as POLAR, and it’s happy to hang out on the surface. 

The -OH also opens up a lot of opportunities for “post-translational modification.” Translation is the process whereby individual amino acids are linked up into a chain (based off of the genetic instructions contained in the messenger RNA (mRNA) copy of the DNA gene for a protein). So post-translational modification just means that you make a change to a protein after you’ve made it. A common PTM threonine undergoes is phosphorylation, in which phosphate groups (central phosphorus surrounded by oxygens) are added. It can have big effects…

jargon watch: regulatory PTMs like phosphorylation usually occur after the protein is folded, but “PTM” can refer to any change that is made after the amino acid has been added to the polypeptide chain – the modifications may be added before/during the folding process. Now back to the phosphorylation story…

Ever try to pack a suitcase and get everything to fit perfectly but then you buy one more item? And then you have to shift things around to accommodate it? The same thing occurs with proteins. When the protein’s initially made (translated) it “packs itself up” nice and neatly by folding into the shape that maximizes beneficial interactions & minimizes unfavorable ones. But then other molecules can add items after the fact (post-translationally) which you have to pack in too. In the case of phosphorylation, these “extra items” are big, bulky, and negatively charged. 

In order to accommodate these phosphate groups, a protein may need to change its shape and/or binding sites, which could affect its functioning and interactions. These changes can be subtle (e.g. just some slight shifting around the modified site), or dramatic; because all the protein’s amino acids are linked together, changes in one can have a “ripple effect.” So a change at one site on the protein can impact the structure of the protein at a distant site on the protein; this is called an “allosteric effect” (allo- means other and -steric refers to the spatial arrangement of atoms). Even small structural changes can have biochemical consequences, altering how the protein functions and/or interacts with other molecules. So phosphorylation can do things like turn enzymes “on” or “off” 

But how/why does threonine get phosphorylated? Let’s look closer at that part that makes Thr different from Val: that alcohol (yup, turns out having some alcohol can make amino acids act a lot differently too…)

The O of -OH (and even more so of its deprotonated form, O⁻) is NUCLEOPHILIC – It has lone pairs of e⁻ & is really looking for some positivity (which it can find in the nucleus of another atom, since that’s where the positively-charged protons hang out). One way to get this positivity is to attack an ELECTROPHILIC atom (something that wants electrons) and share its “extra” e⁻ with it, forming a covalent bond. So threonine has the potential to be COVALENTLY MODIFIED.

One such electrophilic atom is central phosphorus (P) in phosphate (PO₄³⁻). Electrophilic things are often positive (which is why they want negative electrons), so phosphate (with its concentrated negative charge) may not seem like an obvious choice….  But that negative charge is NOT fairly distributed. Instead the ELECTRONEGATIVE (e⁻ hogging) O’s pull e⁻ density away from P, leaving the P partly positive, and, since it’s not getting the electrons it want from the O’s it’s connected to, it’s happy to swap for better alternatives….

One place you’ll find phosphate “on the dating scene” is Adenosine TriPhosphate (ATP), which, as the name suggests, has 3 phosphates. Not only does this offer 3 electrophilic centers, these centers are “extra desperate” to leave their current situation because, while opposites attract (making the partly-positive P attractive to the partly-negative O of threonine), like charges repel. So when you stick 3 phosphates next to each other, it takes a lot of energy just to keep them together. It’s kinda like clamping a spring – when you let go you release energy and if you capture this energy you can use it to do things.

A lone pair of electrons from O can attack the last P in the line (the γ (gamma) P). The P likes having these electrons, but it doesn’t like that it now has has too many bonds, so it breaks its bond to the O connecting it to rest of ATP -> phosphoryl group (PO₃²⁻) transferred from ATP to alcohol to give you a PHOSPHOPROTEIN!

This transfer is catalyzed by a type of protein enzyme (reaction mediator/speeder-upper) called PROTEIN KINASES & it can be “undone” by PHOSPHATASES, thus offering a reversible form of post-translational modification. 

We’ve been talking a lot over the past few days about etymology (where words come from) with regards to amino acid names, but enzyme names can have cool meanings too. “Kinase” comes from word for “move” because kinases move phosphoryl groups. Note:Another class of enzymes, phosphorylases, add “inorganic” phosphate (Pi) directly (not from ATP).

Threonine is not the only amino acid that can be phosphorylated. The most commonly phosphorylated amino acid is serine (Ser, S), which is like threonine but without the methyl group (so Ser is just -(CH₂)-OH). Tyrosine can also be phosphorylated, but unlike Ser & Thr which are similarish in size and thus can often be worked on by the same kinases/phosphatases (Ser/Thr kinases & phosphatases), Tyr is bulky & usually requires different enzymes. 

note: Histidine (His, H) can also be phosphorylated (on one of the ring’s N’s) and this is really common and important for bacterial signaling pathways. It also occurs in mammals, but it’s really hard to study because it’s not very stable, so scientists aren’t quite sure how widespread and important it is, although tools are being developed to study it better, and it’s an active field of research 

Speaking of research and discovering things, now I I want to tell you the story of how we know what we know about threonine. For much of this, we have its discoverer, an American biochemist named William Rose, and his grad students to thank. Rose determined that humans can’t make threonine from scratch and thus must eat it “pre-made” (i.e. threonine is essential). He figured this out by feeding graduate student volunteers strict diets consisting of all the other amino acids (and sugars, fats, vitamins, etc.) with and without threonine and then measuring nitrogen levels in their waste. There was likely a slight panic among the volunteers when another laboratory reported that threonine deficiency lowered sperm count, but this theory was later counted out. 

But I’m getting ahead of myself. Rose started with rats. And he wasn’t the first to do something similar. In the 1910s, Osoborne & Mendel fed rats diets with restricted protein sources. If they fed the rats protein sources deficient in lysine, the rats would stop growing until you added back lysine. Deprive them of tryptophan too, and they’d die unless you added that back in. They determined these amino acids to be “essential”

Starting in the 1930s, American biochemist William C. Rose decided to do things more controlledly – he synthesized the amino acids or purified them himself, then he mixed & matched, so he knew exactly what was in there. He modeled what he gave the rats based on the amino acid content of the protein casein (a major protein in milk). But even though casein was good enough (if you added histidine), this synthetic mix wasn’t. So he knew that he was missing something in his synthetic recreation – another amino acid.

Therefore, Rose set out to find what he calls the “new essential” which sounds like a beauty care product. But easy, breezy, it was not (though the end results were beautiful…) Starting from 12kg of the protein fibrin, he used acid to split apart the amino acids & then he used tons and tons of selective extraction techniques (remove things based on their ability/inability to dissolve in various solutions) to remove the known amino acids. And then he set to work figuring out what else was left that, when added to the known stuff, would allow the rats to thrive. 

You can tell the work (one step of which involved extracting 17 times with 40L portions of butyl alcohol) is hard when scientists tell it like it is in their papers. As Rose, McCoy, & Meyer state in their classic 1935 paper, “no procedure has been found which is not exceeding laborious” 

But triumph they did! From that initial 12kg of protein they were able to isolate ~4.5g, so ~0.6g per kg of that fibrin protein he started with. And in pretty crystal form! They triumphantly (and deservedly) declare, “The data demonstrate conclusively that the crystalline compound is the new essential we have been endeavoring to isolate for several years. Furthermore, the experiments recorded in Chart I represent the first successful efforts to induce growth in animals upon diets carrying synthetic mixtures of highly purified amino acids in place of proteins.” YAY!

But that was hardly the end… they still had to figure out what it was chemically. So they did a lot a lot more chemistry experiments involving things like seeing what it would react with, trying to make other things with it and characterizing those things too, etc. And they were able to determine it to be α-amino-β-hydroxybutyric acid. They knew that the side chain was a butyric acid group BUT they didn’t know what stuck off which way. 

The other day we talked about stereoisometry, where the same atoms can connect in different ways 3D-space wise if you have 4 different groups attached to a carbon (we call such places chiral carbons or steric centers). More here:

Most amino acids only have a single steric center, the α (alpha) carbon in amino acids, which is that central hub that the side chain sticks off of. So they only have 2 stereoisomers (designated L & D, with L being the one our bodies use). But threonine has a second one in its side chain, so it has 4 possible stereoisomers. And while they could tell it was L, they couldn’t tell which L. The answer to this would come later – when H.E. Carter synthesized all 4 options and showed that the natural one corresponded to (2S,3R)-2-amino-3-hydroxybutanoic acid. Isoleucine is the only other one with a second steric center in case you were wondering…

But anyways… so now they’ve figured out that threonine was essential, and now that they had the full set of amino acids they could make a totally “artificial” diet protein-wise. So they could exclude one of them at a time and see what happened. And they could see just how much of each one was required for rat healthfulness. 

But were rats really “human-like” enough? He next turned to dogs and got similar results. But dogs still aren’t human… If he really wanted to know whether threonine was essential (and whether the other ones he thought were were) he would need to turn to human “lab rats.”  And that was where the grad student volunteers came in in the 1940s. (Apparently no one told them that in grad school there are tons of better ways to get free food…) Thankfully they only experienced some minor symptoms, like irritability, fatigue, and loss of appetite. And only when certain amino acids were withheld: isoleucine, leucine, tryptophan, lysine, methionine, phenylalanine, threonine, and valine. Thus, Rose was able to figure out that these amino acids are “essential” whereas the rest can be made from other things in our bodies (and he fed the students normal food again so they perked up).

for more information: 

how does it measure up?
coded for by: ACU, ACC, ACA, and ACG
empirical formula: C4H9NO3
molar mass: 119.120 g·mol−1

This post is part of my weekly “broadcasts from the bench” for The International Union of Biochemistry and Molecular Biology. Be sure to follow the IUBMB if you’re interested in biochemistry! They’re a really great international organization for biochemistry.

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉

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