Move over Kelloggs, this “K” is way more special than your cereal! Why? For one, the amino acid (protein letter) Lysine (Lys, K) seems to have a random abbreviation… but that’s just cuz the more “logical” ones were taken, & K was closest to L in alphabet! That’s not what makes lysine really special… It can exist in positive-charged or neutral states – cool but not unique (arginine and histidine have that superpower too). Nope – what really makes lysine special is its ability to form “Schiff bases” that can provide reversible yet strong linkages between proteins and other things. Those other things can be modifying groups like ubiquitin (which can tag proteins for degradation). or helper molecules (cofactors) that help it speed up (catalyze) many a biochemical reaction. So let’s take a look at lysine in action! 

It’s Day 12 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 Lysine!

But before we get into examining the special part, let’s look a little closer at the generic part. All amino acids have that nitrogen (N) attached to the central, alpha carbon (Ca) and its this nitrogen that gives them their “alpha-amino” designation. “Amine” is a name we give to nitrogen attached to things and we can further classify amines as primary (attached through a single bond to a single carbon-containing group (abbreviated C)), secondary (2 links to carbon), tertiary (3 links), or quaternary (4 links to C). 

In their free state, all amino acids (except for proline because its side chain wraps around and binds its N) are “primary amines” – they have primary α-amino groups. When amino acids join up through peptide bonds, that N gets attached to a C from its neighbor, so it becomes a secondary amine. 

Lysine has an *additional* primary amino group at the end of its side chain. Speaking of that side chain, Lys’s side chain is a “butylamine.” The “butyl” refers to the 4 methylene (-CH₂) linker between the Ca it sticks off from and that end amino group. The atoms of K’s side chain are sometimes given numbers starting w/the carboxylate C of the backbone (in this method, the amino group is attached to C₆) & sometimes, instead of numbers, they’re given Greek letters starting with the backbone’s C (in this method, the amino group is attached to C𝜀). Because of this Greek lettering scheme, lysine is often described as having an ε-amino group. 

Under physiological (normal bodily) conditions, the nitrogen of this amino group is attached to as many hydrogen atoms as possible (i.e. it is fully protonated). For a primary amino group, this means being bound to 3 H’s, and it leads to a positive charge (-NH₃⁺). Why?

Like all the bonds we’ve discussed so far in this post, these N-H bonds are “covalent bonds” meaning they involve atoms sharing pairs of negatively-charged subatomic particles called electrons. These electrons whizz around in “electron clouds” around a dense central core called the atomic nucleus where positively-charged protons (with some gluing together help from neutral neutrons) are tasked with reigning them in.  When the # of protons = # of neutrons you get an overall neutral molecule. Since electrons can wander a bit, you might get an unequal distribution of the charge over the molecule (i.e. some partly positive parts and some partly negative parts) but there’s no net charge. But if the # of electrons > # of protons you get a negatively-charged (anionic) molecule and if # of electrons < # of protons you get a positively-charged (cationic molecule).

If you look at a primary amine linked to only 2 hydrogens, you’ll likely see a pair of dots above the N. Those dots represent a “lone pair” of electrons which aren’t involved in any bonding. Since a single bond only involves 2 electrons, this lone pair is capable of bonding to something which doesn’t have any electrons of its own to offer up. Therefore, it can bind a proton (H⁺) and acquire that proton’s charge. (Hydrogens only have a single electron to begin with, so if they leave without it, they’re just left with a single proton, so we often call “H⁺” a proton). So when lysine’s side chain is in its fully protonated state, it’s positively-charged (-NH₃⁺).

Nitrogen might seem really generous offering to share its lone pair with that proton. But it’s not a fair sharer… Nitrogen is an electron hog (highly electronegative atom) so it pulls electrons that it shares with hydrogens away from the hydrogens. This can lead the hydrogen to “give up” and leave without its electron (i.e. leave as a proton). Thus we say lysine’s side chain can exist in fully protonated (-NH₃⁺) and deprotonated (-NH₂) states. 

This puts lysine in same category as the amino acids arginine (Arg, R) & histidine (His, H). (We haven’t gotten to these yet, but we will!) All 3 of these can be + charged (cationic) or neutral depending on their protonation state, and which state they’re in depends on how many free protons are around to take. If there are lots up for grabs, the amino acids will likely be protonated, but if protons are harder to find, they’re less likely to bind!

A measure of free proton availability is pH. It’s an inverse log scale, so the more protons there are, the lower the pH (more acidic conditions) and the more likely lysine is to be protonated. The pKa is the pH at which half of something will be protonated (at lower pH, more than half will be and at higher pH, less will be). The pKa for lysine’s ε-amino group is ~10.5. Since bodily pH (outside of special compartments) is ~7.4, lysine is therefore positively-charged most of the time. 

Something that confused me for a long time (and which I still often have to think twice about) is that Lysine (as well as His & Arg) is often called “basic.” This doesn’t mean it’s simple – instead it means that it often acts like a base in the acid/base definition where an acid is something that donates a proton and a base is something that takes a proton. Under normal bodily conditions, lysine has taken a proton, so it’s *already* acted as a base (and now can act as an acid…)

But Lysine’s acid/base status is “negotiable” – the protonated form can be convinced to give up that proton, which leaves Lys’ N with a lone pair of electrons & makes it both a good BASE & a NUCLEOPHILE. A “nucleophile” is something that seeks out ➕ charge. It gets its name because it “likes nuclei” because they contain ➕ protons. “Base” is a term we give to a nucleophile when it steals a proton as just a handoff. BUT a nucleophile can also form bonds to things other than H. And this is a common way of linking together molecules. more here: http://bit.ly/nucleophilefiles 

Nucleophiles are often ➖ &/or contain lone pairs (you can remember this by picturing “u” in “nucleophile” as a smiley face w/ lone pair eyes). The opposite of a nucleophile is an ELECTROPHILE (something which “loves e⁻“), which are often➕ (or partially ➕). Opposite charges attract, so NUCLEOPHILES react w/ELECTROPHILES, often through nucleophilic substitution reactions. more here: http://bit.ly/sn1vssn2 

This strong nucleophilicity potential makes K very special in many enzymes. Enzymes are reaction speeder-uppers (catalysts) – usually proteins, sometimes protein/RNA (like ribosomes) and sometimes just RNA. They’re able to help reactions happen by bringing reacting molecules together, holding them in place, providing the right environment, etc. http://bit.ly/enzymecatalysis 

Often that “right environment” involves an optimal pH. We normally think of pH at the more global scale, but you can also think of pockets of enzymes where reactions occur (active sites) as having a “local pH” which is more easily skewed (think of a national poll vs. neighborhood polls). Just like a candidate campaigning or not campaigning in a district can skew the polls, lysine can skew the pH – it can act as an acid or base, taking & donating H⁺ to speed up reactions.

In addition to changing the local environment, lysine can interact directly with other molecules. And thanks to its special amino group – as we’ll look at more below – it can do so with strong, covalent, linkages. This allows it to hold enzymatic helpers (small molecules called cofactors which often come from vitamins) in place.

This is possible because Lys can form SCHIFF BASE intermediates. In these reactions, lysine’s end N “replaces” O in a carbonyl (C=O) group in an “aldimine” linkage (so you go from thing1-C=O + NofLys-thing2 to thing1-C=NofLys-thing2. jargon note: that N of Lys is now part of a secondary amino group, and since there’s a C=N double bond, we can call an imine.

This “Schiff base” can hold cofactors in active sites. But the really cool thing about it is that it’s strong but *reversible*. The C is happy to be bonded to O or to N so you can keep swapping out what it’s linked up to. Therefore, you often you get a scheme like:

thing1-C=O + NofLys-thing2 to thing1-C=NofLys-thing2

thing1-C=NofLys-thing2 + thing3-C=N -> thing1-C=N-thing3 + thing2

voila – you’ve just done a handoff! This is useful in lots and lots of reactions in your body including a type of reaction we see over and over in amino acid breakdown (catabolism) – transamination. As we saw, even the amino acids that don’t have nitrogen in their side chain still have it in their backbone, and that N has to get removed if you want to make non-nitrogen-containing things like sugars & fats from the amino acid parts. So usually the first step in breaking down any amino acid is removing its amino group. And this is done in a “transamination” reaction that passes the amino group off to α-ketoglutarate (an intermediate in the sugar breakdown process called glycolysis). But it’s not a direct handoff – instead, that N is first handed off to a cofactor in a transaminating enzyme (transaminase).

One of the main transaminases is alanine aminotransferase (ALT) and it uses a cofactor called pyridoxal phosphate (PLP), which is made from vitamin B6 (pyridoxine). As the “al” in the name hints at, it’s an aldehyde, which means it has a -C-(C=O)-H group. And when there’s a carbonyl there’s a place for lysine to make a deal! Lysine forms a Schiff base with the PLP, “replacing” the carbonyl with its nitrogen (and kicking off a water). 

So 

free PLP + enzyme -> enzyme-PLP

Now, a free amino acid comes along to get transaminated. It replaces the enzyme-PLP bond with an amino acid-PLP bond in an imine swap. 

So

enzyme-PLP + free amino acid -> amino acid-PLP + enzyme

At this point the amino acid-PLP is not covalently-bound to the enzyme, but there are also weaker, non-covalent interactions, so it sticks around – long enough for….  water to come along and split it up (hydrolyze it) releasing an a-keto acid.

But you’re not done yet. Now you have that nitrogen stuck on the PLP – we call this intermediate pyradoxamine phosphate (PMP). If you want the PLP to be “usable” again, you’re gonna need to get rid of that nitrogen. So, next, an a-ketoglutarate comes around with its carbonyl and you get another swap 

PMP +  a-ketoglutarate -> PLP + glutamate

The glutamate can then go take that nitrogen off to the urea cycle to remove it.

And now all that’s left is for the enzyme’s lysine to reattach itself to the PLP – and it’s ready to do it all again!

med note: You have a lot of ALT inside your cells, especially in your liver. It gets released when cells die – so doctors measure it in blood to detect diseases like liver problems. 

The ability to form these special bonds opens up a lot of possibilities for post-translational modifications. “Post-translational” refers to changes made to amino acids after they’re added to a growing chain during the process of protein-making (translation). There are 20 *common* amino acids, but there are also *uncommon* ones, some of which come from modification of K. 

The other day we looked at collagen when we were talking about how it has a cool triple helix structure with lots of proline and glycine. http://bit.ly/prolinestuff 

Collagen also has 5-hydroxylsine (HyLys). This is lysine with a hydroxyl (-OH) group added on. HyLys creates strong covalent crosslinks between strands of collagen, which helps give collagen tensile strength (ability to be pulled without breaking) greater than that of steel wire of equal cross section.

Other, reversible, modifications include methylation (1, 2, or all 3 of the amino group’s H’s can be replaced by methyl (-CH₃)) & acetylation (H replaced by “acetyl” (carbonyl attached to a methyl)). These modifications  have a variety of functional consequences, including altering gene expression. 

In order to fit within our cell’s nuclei, DNA is compacted by coiling around protein complexes called HISTONES ➿.  more here: http://bit.ly/nucleicacids2 

These proteins have “tails” containing lots of K &  methylation & acetylation of these K contribute to a “histone code” that tells a cell when to express certain genes. It’s not quite this simple, but acetylation helps “loosen up” regions, whereas methylation tightens things up. This can make regions more or less accessible for transcription (making messenger RNA (mRNA) copies of a gene getting made which are later read by the ribosomes which make proteins based on their instructions). 

Lysine can also be used to send other signals “further down the line” once proteins are actually made. Sometimes your cells have proteins that are either defective or just not wanted anymore. Lysine offers a linker for Ubiquitin (Ub) a small protein that can get added to other proteins to flag them for degradation. Ub’s recognized by a protein chewing complex called the proteasome, which prevents toxic proteins from building up and allows the amino acids to be recycled. more here: https://bit.ly/ubiquitinylation

Also, mRNA gets a generic “poly-A” tail added. Here we’re talking A as in the RNA letter adenine, not the protein letter alanine! In fact, if the ribosome blows through the stop codon or there isn’t one thanks to a mutation these As get read by the ribosome. And AAA (and AAG) spell lysine! So a bunch of lysines get added and then those can be ubiquitinylated so the cell can degrade these problematic peptides! (this process is called nonstop decay). More here: https://bit.ly/rqcontrol

Now for some historical notes…

Where was lysine first found? It was discovered in 1889 by German chemist Ferdinand Heinrich Edmund Dreschel in 1189 in the milk protein casein. He gave it the name lysine (first lysatine) from the Greek word for “loosing” because in the process of isolating it, he accomplished the first time urea was obtained from protein by hydrolysis. https://doi.org/10.1021/cr60033a001  

Where will you find it? Well, charged things like to hang out with oppositely-charged things – even if the charges are only partial. And thanks to oxygen hogging hydrogens’ electrons, water has partially charged regions (we call such molecules polar), so the tail of lysine’s tail likes water (is hydrophilic). But that linker region is a long stretch of H & C which share electrons pretty fairly and thus are neutral everywhere and “boring” to the water, which excludes these nonpolar regions, choosing to bind to things it likes instead. So lysine is characterized as AMPHIPHILIC (have polar & nonpolar regions) and prefers to be on the protein surface but can have linkers more interior. It can can form “salt bridges” (➕/➖bonds, aka ionic bonds) which help maintain protein’s 3D structure & mediate protein-protein & protein-nucleic acid (DNA/RNA) interactions.

Lysine is classified as ESSENTIAL (body can’t make it) & KETOGENIC (its breakdown products make ketone bodies which can be made into lipids). It’s essentialness was discovered in 1914 by Osborne & Mendel – and this was part of the work that inspired William Rose to do those grad student studies that led to finding the last of the 20 common amino acids, threonine. More here: http://bit.ly/threoninetale

Final notes on how it measures up:

systematic IUPAC name: (2S)-2,6-Diaminohexanoic acid
coded for by: AAA, AAG
chemical formula: C6H14N2O2
molar mass: 146.190 g·mol−1

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

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