Today I discovered that my timer is magnetic. And I stuck it above my bench while doing an enzyme assay and it made me smile. And enzymes make me smile all the time. The word “enzyme” literally means in (“en”) “leaven” (“zyme”) – but, for me enzyme means more like “in heaven” – I love these things! So I hope you have some time to discuss the ENZYME! (and thanks to enzymes, reactions take less time!) Enzymes (which are usually proteins – sometimes protein/RNA combos, or just RNA) got their name in 1878 from Friedrich Whilhelm Kuhne who used the term to something that was speeding up bread-rising-reactions. But enzymes can speed up all types of biochemical reactions – by 5-17 orders of magnitude! So like quadrillions of times! 🤯
One of the speediest enzymes known is carbonic anhydrase, which can combine CO₂ with water to make carbonic acid – or do the reverse – 1 MILLION TIMES PER SECOND! Which is 10 million times faster than it would happen on its own. And this speed is critical because it allows our bodies to get CO₂ out of our tissues and helps maintain pH balance (how acid-y your blood and cells are). It might seem like magic – but it’s just really cool science! (note: most enzyme turnover rates aren’t this high!) So – what’s the deal with enzymes?
Enzymes make me happy so it seems only fitting to talk in terms of rainbows, right? Enzymes give you an easier path to the pot of gold on the other side! They speed up chemical reactions by lowering the energy you have to put in (ACTIVATION ENERGY, ΔG⧧) to get past an ACTIVATION BARRIER (a TRANSITION STATE (⧧) in which the reactants are very uncomfortable) before they can react to form products. Don’t worry – I’ll explain this all (and hopefully understandably so!)
Imagine you’re on the “short side” of a candy-cane shaped rainbow. At the bottom of the rainbow on the other side is a pot of gold – but in order to get to it the usual way you have to climb to the top of the rainbow before you can slide down. Even though you’ll be happier when you get to that pot of gold, you have to trudge up the rainbow hill first – and sometimes it just doesn’t feel worth it – especially if you don’t know that there’s a pot of gold on the other side!
Similarly, when molecules interact and change by breaking and forming bonds, etc, they don’t know that there’s something better they’re going towards. They just know they’re “uncomfortable” with their current conditions and so they react in ways that make them more “comfortable” – enzymes provide an alternative, more comfortable path to the pot of gold! It’s a 2-way street, so enzymes also make it easier to go back – but the enzyme doesn’t change which way the reaction likes to go – so if the pot of gold were at the top of a steep rainbow, having a direct route to the top might make the trip a little faster, but it won’t make it favorable!
The “rainbow” in biochemical reactions is a REACTION COORDINATE DIAGRAM which is basically a molecular uncomfortableness curve. This “uncomfortableness” I’m talking about is more conventionally called FREE ENERGY and reactions are energetically favorable if the products have LESS free energy than the reactants.
Imagine you’re getting a picture taken. The more uncomfortable a position is, the harder it is to hold still in it, so the more energy you have to put into maintaining that cheesy smile. When molecules are really “uncomfortable” in their current state we say they have high free energy. And when it comes to this type of free energy, a guy named Gibb gave us a lot of what we know, so we call it Gibb’s free energy and abbreviate it G.
So, uncomfortable molecule -> high G
At the other extreme are the “candid poses” – when molecules are comfy – they don’t have to spend as much energy just “posing” (so they have low free energy, low G) so they can relax and just do their own thing.
So, comfortable molecule -> low G
And the difference between the high G & low G states is ΔG (Δ is pronounced “delta” and it means “change in”)
Wireless network providers are always bragging about their “high G” networks – but in biochemistry, low G is how molecules like to be! So they’ll act and interact in ways that get them to that low G state (hence FAVORABLE interactions have a NEGATIVE ΔG)
But getting there isn’t always easy – in fact, they often have to get more uncomfortable before they get comfortable – before reaching the low-G product “ground state” they have to pass through an uncomfortable TRANSITION STATE. The energy required to get to that transition state (through the ACTIVATION BARRIER) is the ACTIVATION ENERGY (ΔG⧧). Catalysts work by lowering this barrier, so less activation energy is required for the reaction to proceed – thus your reaction can occur at a much higher speed! But IN BOTH DIRECTIONS! (they can’t make you happier in a stiff pose but they can make it easier for you to choose to pose or not). Catalysts can’t change the overall change in free energy, ΔG because that’s one of those “state properties” – it depends ONLY on initial & final “states” NOT path taken to get there
You can find all kinds of catalysts all over the place – you might have heard of “catalytic converters” in cars – the catalysts you find in your body (biological catalysts) we call ENZYMES. Enzymes are usually proteins, but can also be protein/RNA complexes or RNA alone (RNA deserves more credit…) & they can also include helper molecules called “cofactors.” For today’s talk we’ll assume we’re talking protein. In enzyme-catalyzed reactions, the reactants are often called SUBSTRATES (S) & they bind to a special “pocket” on the enzyme (E) called the ACTIVE SITE (aka catalytic site) which provides an ideal environment for the reaction to occur.
There are lots of different types of reactions they can speed up – everything from RNA writing (transcription) to ATP-making to DNA breaking! They can do what they do in my ways including by simply getting molecules (which are swimming around happily) to stay still long enough to react and holding them down in a position that’s ideal for that reaction to occur. This can often involve bringing molecules together in the “right orientations” (what are the chances molecules would happen to collide with one another in just the right way?)
To illustrate in a less-rainbow-y, more realistic (but even more incredible!) way, let’s look at that last one – DNA “breaking” because a common type of enzyme that’s used a lot in biochemistry and molecular biology in a DNA breaker!
Restriction enzymes (site-specific endonucleases) are proteins that recognize specific sequences in DNA and cut them, making them really useful for things like molecular cloning where you can take DNA from one place and stick it somewhere else. Restriction enzymes are also useful for checking to see if a sequence is present (“Restriction Fragment Length Polymorphism” (RFLP) used to be used a lot by CSI people and paternity testers and geneticists trying to track down diseases – now that stuff’s mostly done by PCR based methods but we still use them in a similar manner to check if the DNA we were trying to put somewhere (like a gene we’re putting in a circular piece of DNA called a plasmid) actually got there.
Restriction enzymes take as a substrate (S) a longer piece of DNA (and a water molecule), and the products (P) are 2 shorter pieces of DNA (this is assuming you’re starting with linear DNA – if you cut a circular piece of DNA like a plasmid 1 time you still have 1 piece, but that piece has a lot more freedom to move around.
Molecules like to have freedom to move around – the more different ways they can move, the more ENTROPY they have. Entropy is often described as randomness or disorder – and it fits into the whole “many ways to move” thing because if something’s moving around a lot each time you look it’s likely to be in a slightly different position. Entropy is directly related to free energy, as we looked at in my post on thermodynamics http://bit.ly/2lVjuuc
In the equation ΔG = ΔH-TΔS, entropy is the S (don’t confuse this with substrate!). H is enthalpy which has to do with bond energy stuff & T is temperature (in Kelvin (K)). As you can see, entropy gets subtracted, so the higher the entropy difference, the more negative (thus more favorable) the reaction. So reactions often proceed in a way that leads to greater entropy (more molecular freedom).
A long piece of DNA is kinda like a more than three-legged, three-legged-race. The outer legs have more freedom to move than the inner legs and the more racers are in the line, the harder it is for the line to move. Each time a racer links up to another racer, the outer racer looses a little bit of her freedom (and so does the newly added racer). And each time a racer breaks off, they get that freedom back.
So, why doesn’t DNA just randomly split up? Because the activation barrier’s way too high! As the songs will tell you, breaking up is hard to do – even if the end result is happier people – and even if those “happier people” are actually “more comfortable” molecules – (molecules with lower free energies). So they often need help in order for the reaction to proceed at any appreciable speed.
Endonucleases hold the DNA in a more readily cleavable position and stabilize the awkward transition states – including through the use of a metal cation (often magnesium, Mg2+) – the cation’s positive charge helps coordinate with the DNA’s negative charge and hold things just right while the protein helps a water molecule attack the bond between the DNA letters (nucleotides), so they split
If the molecules are MUCH happier apart than they are together, they’re unlikely to get back together even if they meet each other in an ideal setting, like the grips of an enzyme active site (nothing screams ambiance like a pocket in a protein, right?) But if the molecules are more “wishy-washy” about whether they really want to be separated, they can join back together – and the same enzyme makes this easier too! Because that rainbow of a reaction energy diagram is a 2-way street! And the easier route enzymes provide is easier in both directions.
It’s kinda like a TV show where a married couple clearly needs a divorce but the conditions never seem right. it’s like an enzyme guides them to city hall to get that divorce. But the enzyme can also guide the separated couple to city hall to get married. If the people really don’t like each other, they’re not going to get married even if they keep meeting at city hall. But if they do still love each other and want to be together, and they keep meeting under these ideal conditions, they might get back together.
When endonucleases cut DNA, the pieces are way happier apart so they’re super unlikely to get back together – that requires the help of another enzyme, DNA ligase.
In the pics I give a couple specific examples of how enzymes catalyze different reactions.
The general reaction scheme for an enzyme-catalyzed reaction is where you have a single enzyme (E) binding a single substrate (S) (reversibly, hence the ⇌, and turning it into a product, P.
E + S ⇌ E + P
Along the way, you go through a couple transition states – you initially have E bound to S (ES) then S changes to P but it’s still bound to E (so EP) before it gets released (E + P)
So overall, you can write
E + S ⇌ ES ⇌ EP ⇌ E + P
You can see that S changed to P but the E is still E! Like all catalysts, it doesn’t get used up. This is one important quality of ALL catalysts – they are NOT USED UP in the process – they can keep going over and over and over. Because even while they’re changing other things, they’re not getting permanently changed (the enzymes often change shape temporarily (and sometimes even hold onto product parts along the way, but they always get returned back to where they began)
Another property of all catalysts is that marriage/divorce thing – they are “unbiased” – meaning that they speed up both the forward and reverse rates of a reaction. So they can’t make a reaction that doesn’t want to happen happen. But they can make a reaction that does want to happen happen faster. Often you have reactions that are more “wishy-washy” – if there’s not a big free energy difference between the reactants and the products, the reaction can easily go backwards and forwards, and the enzyme will happily help in either direction. But the enzyme does NOT change the overall ΔG.
Another important property about *enzymes* (which is not true for all catalysts) is that they’re really SPECIFIC. This allows restriction enzymes to only cut at certain sequences and it has to do in large part with how the substrate matches the active site of the enzyme (in this case how the DNA bases fit (or don’t fit) into the pocket of the endonuclease) – shape-wise. charge-wise, etc.
In order for the reaction to be catalyzed, the active site needs to be the right “shape” & have the right “lining” for energy-providing interactions to occur. Shape of the active site is determined by the protein’s sequence of amino acid “letters” (1° structure) & how their backbones (generic) & side chains (unique for each amino acid) interact (2° & 3° structure) – and the “lining” depends on which side chains stick out into the pocket. Thanks to years and years of evolutionary tinkering, different enzymes can be found that can act specifically on all sorts of different things – and speed up reactions WAYYYY better than any human-made machines can – also I guess technically a lot of these enzymes ARE human-made!
⚠️ Bumbling biochemist PSA: E-S interactions are often analogized to a lock & key, implying that E’s active site is a perfect match for S. THIS IS NOT TRUE!!!!! INSTEAD, the active site is a match for the TRANSITION STATE – and it’s not a *perfect* match for that either! ⚠️
At first this can seem confusing. Don’t you want S to bind? Yes – so while the active site isn’t a *perfect* fit for S, it needs to be an OK fit, so you get initial binding. BUT you don’t want it to be *too* good a fit. If you stabilize S, you decrease S’s free energy (G) which “raises” the activation barrier in relation. Basically, if you make S too “comfortable” it’ll have less drive to react. If things are good, why change?
INSTEAD, you want to stabilize the “tipping point” where S has to “take the plunge” & commit itself to becoming P – yup, I’m talking about that TRANSITION STATE (peak of barrier). Once it binds, S shifts around to try to get comfortable. And it finds that the most comfortable position is also the condition most favorable for the reaction!
And the enzyme itself can shift around too – I told you it wasn’t just “lock and key!” – we call protein-shifting “conformational change” and when it happens to better hug a substrate we call it INDUCED FIT. These changes can be subtle (such as rotating an amino acid into place) or dramatic (such as moving a chunk of the enzyme to “clamp down” around the bound S).
The repositioning of substrate and enzyme involves the formation of LOTS of individually weak interactions between E & S. Each time one of these interactions form, the molecules get a little comfier, so small bursts of energy are released. And these small bursts sum together to give you an (often very large) negative binding energy (ΔGB) which serves as an “energy payment” which offsets the barrier “cost” ΔG⧧. So the net ΔG⧧ is lower and the reaction is faster.
There are 7 major classes of enzymes (the 7th was only added in August 2018), and there are bunch of subclasses, and they’re numbered according to an international standard – too many names like trypsin that can trip people up cuz they don’t tell you anything about what it actually does (trypsin is a protease – one of those protein cutters – and it falls into class 3 (Hydrolases) which catalyze “hydrolysis reactions” – basically they use water to help split things (like cut proteins – or DNA! The restriction enzymes fall in this class too)
The enzyme classification scheme was established by the precursor to today’s IUBMB – and the IUBMB still is in charge of it – I’m gonna do a more detailed post on classes and some examples later, but here’s a quick overview to tide you over.
The other classes are
- oxidoreductases: catalyze redox reactions (electron transfers)
- transferases: catalyze group transfer
- hydrolases: seen those
- lyases: add or remove things to make double bonds
- isomerases: help shift groups around within a molecule (like moving an oxygen 1 carbon over)
- ligases: use ATP to join together 2 things (like that DNA ligase I mentioned earlier)
- translocases: help move things across or within membranes
Regardless of class, they can mix n’ match different strategies for lowering the activation barrier. Some common problems they face and solutions they provide:
Problem: entropy – molecules moving all around a lot lowers the probability they’ll collide productively
Solution: enzymes bring em together & “clamp em down” (and in the right orientation)
Problem: solvation shell – interactions w/water allow the substrate to dissolve BUT can also “mask” regions involved in reaction
Solution: enzymes remove that water coat but keep the substrate soluble by “replacing” bonds to water w/bonds to itself
Problem: substrates often have to be distorted (stretched bonds etc.)
Solution: enzymes STABILIZE awkward TRANSITION STATES
Problem: reactions may require unstably charged intermediates
Solution: enzymes LOAN protons (H⁺) or electrons (e⁻) (they get them back – catalysts DON’T get used up ♻️). The electron-loaning is often done with the help of metals.
Metals are just one type of “cofactor” enzymes can use as helper molecules – we call enzymes with metal helpers METALLOENZYMES. Other helpers include bigger organic (carbon-containing) groups we call coenzymes like that NAD we looked at last week. If those helper molecules are covalently bound (those strong bonds that include electron sharing) we call the helper a prosthetic group. When an enzyme has a helper, we call the enzyme/helper combo a HOLOENZYME and the enzyme alone (no helper) the APOENZYME.
more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0