We can classify proteases/peptidases in a couple of ways…⠀
- where they cut: “Endo”protease/peptidase refers to ones that cut in the middle of peptide chains and exoproteases/peptidases chew off the ends. ⠀
- what cutting mechanism they use (what gives these scissors their blade): ⠀
- pepsin is an example of an aspartic protease - it uses an Aspartate (Asp) amino acid residue to help water attack and break a peptide bond. Glutamic proteases work similarly, but they use Glutamate (Glu) instead of Asp. ⠀
- metalloproteases use a metal to help the water out⠀
- In all of those, water is doing the actual work, so there’s no “covalent intermediate” whereby part of the cut protein is stuck to the protease. However, with serine proteases (which I will discuss in the most detail) which use Serine (Ser), there *is* a covelent intermediate. Similarly for cysteine proteases (which use Cys instead of Ser) and threonine proteases which use Thr instead. So it’s protease + peptide → proteasepep + tide → protease + pep + tide (hopefully that’ll make sense later and/or in figs)⠀
- another terminology note that’s easy to get confused about - when we classify proteases as “aspartic proteases” or serine proteases, we’re talking about what amino acid is in the active site of the protease doing the work. We are *not* referring to where on the protein or peptide the protease likes to cut. (i.e. a serine protease doesn’t prefer to cut next to serine).
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Serine proteases all have this conserved catalytic triad of a serine (which directly interacts with the peptide) helped out by a histidine (His, H) & an aspartate (Asp, D), which, through a “charge relay” help get the serine to give up a proton and become more reactive (we’ll get more into this below). Around this conserved core, evolution's “played around” with the surrounding parts, allowing different serine proteases to specialize in cleaving at different spots on proteins, & adapting to work in different environments.⠀
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When serine proteases cut, half of the thing they cut gets “stuck on them” so you need a second cutter to cut it off. So they use a “ping-pong” mechanism. First, the serine gets activated so it cuts the peptide bond. Now half of the peptide’s stuck to the protease & 1/2 is released. But you can’t use the serine to cut off the stuck part because the serine’s what it’s stuck on! So it brings in a water molecule. And it activates it. Then the activated water frees the serine to do it again.⠀
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In order to do all that activating (of serine & water), you need the help of the triad’s other members, His & Asp. You don’t want to activate Serine until go time, so all’s calm until substrate binds to a specificity-determining pocket. This gets the protein to shift a little and this makes Asp’s O- move closer to His, drawing away His’s H, so it’s more willing to steal Ser’s H, giving you an “alkoxide ion” ⠀
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Ser really doesn’t want negative charge, so it goes on the nucleophilic attack, attacking the carbonyl carbon of the peptide bond in the substrate. And this might make Ser happy, but it kinda throws everything else out of whack - carbon’s left with a really unstable tetrahedral (bound to 4 things) intermediate - it’s gonna break - but the key is to break it the “right way” - and this is helped out by a “side-pocket” of the enzyme called the oxyanion hole - it helps stabilize this unstable intermediate so that it splits the right way - breaking the peptide bond between the carbon that was the carbonyl carbon and the nitrogen, with the nitrogen snatching off the proton that His stole from Ser on its way out.⠀
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Congrats - you’ve just cut the peptide & released part. But, before you start celebrating… you still have the other part stuck on Ser… So now we have to get that off. And this is the “slow” part - because we have to get water in and activated. The His is going to do the activating again, just like it did with Ser (remember that the peptide took the proton from His that His took to Ser, so there’s now room for another proton there!). So the nitrogen of His snatches a proton from water, giving you a hydroxyl ion, which for reasons akin to the reasons the alkoxide ion attacked the carbonyl C, will nucleophilically attack the carbonyl carbon. So you get another tetrahedral intermediate that gets broken by Ser’s O taking a proton from His. ⠀
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It sounds like you could make a good kids’ book or song from it…. Ser steals a proton from His that His stole from water to replace the proton that the N-terminus stole from His that His stole from Ser! And with this, the C terminal is released and the enzyme is back to its starting state so it can do it all again.
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