Proteolytic Enzymes

This post involves a love story too, though it's a somewhat different love story.

Meet Nucleophile and Electrophile. Nucleophiles (nucleus-lovers) tend to be attracted to positively-charged things (generally stuff with relatively few electrons), and electrophiles (electron-lovers) tend to be attracted to negatively-charged things (stuff with relatively more electrons):

(Sorry if that was a shitty and/or inaccurate description btw, I don't know how to explain this.)

Occasionally nucleophiles impose themselves (for lack of a better word) on the electrophiles. This process is known as "nucleophilic attack" and is much less brutal than it sounds. A lot of reactions involve such nucleophilic attacks. These include reactions by proteolytic enzymes that cleave peptide bonds.

One type of catalytic reaction is acid catalysis. This is relatively rare in biological systems as it requires very low pHs, and generally biological systems are at a pH of around 7. It's a fairly good place to start though, so let's take a look at how acid catalysis works.

In peptide bonds, there is a carboxyl group and an amino group joined together. The oxygen on the carboxyl group (the carbonyl oxygen) tends to carry a slight negative charge as O is more electronegative (more likely to attract electrons) than C. This makes the O nucleophilic. Hydrogen ions floating around in acid solution are electrophilic- they're just a proton, after all. Hence the carbonyl oxygen attacks the H⁺, sharing some electrons with it, like so:

This results in the breakage of one of the C-O bonds and the formation of an O-H bond. The removal of electrons from the C=O bond also makes the carbon less negatively charged (more positively charged) so that water can come in and attack. At the same time, more crazy attacking stuff happens: namely, the amide group attacks one of the hydrogen atoms on the water, and the C=O bond reforms:

This results in the formation of separate carboxylic acid and amino groups, like so:

Note that the H+ did not change in the reaction. Remember, catalysts speed things up without actually being changed or consumed by the reaction.

General acid catalysis is done using weak acids, rather than strong acids, so general acid catalysis is more likely to be seen in biological systems. The process is pretty similar to acid catalysis, except that the water nucleophilically attacks the carbonyl carbon at the same time that the weak acid protonates the carbonyl oxygen. (Sorry, no pictures, I'm done drawing already.) When the water nucleophilically attacks the carbonyl carbon, the O and one H of the water bind, leaving another H around. This H gets nucleophilically attacked by the amino group, and the C=O reforms.

Metal ion catalysis is similar to general acid catalysis, but with a metal ion rather than a weak acid.

Hydroxide ion catalysis is the opposite of acid catalysis: it occurs at very high pHs. Here you have hydroxide ions floating around in solution. This is kinda similar to the other three above, except no protonation or addition of a metal ion is needed at the beginning. Instead, a hydroxide group directly attacks the carbonyl carbon, and one of the C-O bond breaks to leave the carbonyl oxygen negatively charged. Then the amino group attacks one of the hydrogens on the water, and the C=O reforms (sound familiar)? The loss of the one hydrogen from the water means that the original OH^- group is reformed as well.

Finally, in general base catalysis, the weak base attacks one of the hydrogens on water (forming a weak base bonded to H), and the resulting OH^- group attacks the carbonyl carbon. This results in the breaking of one of the C-O bonds to leave the carbonyl oxygen negatively charged (just like in hydroxide ion catalysis). The amino group then attacks the H bonded to the weak base, and the C=O reforms.

Overall, these catalysis reactions can be summarised in three main steps:

1. Something nucleophilically attacks something else
2. A tetrahedral transition state forms
3. The C-N bond breaks by the amide group nucleophilically attacking a hydrogen, and the C=O bond reforms.

Now, why go on and on about catalysis? That's because proteolytic enzymes use these reactions to catalyse the breakage of bonds.

Proteolytic Enzymes

There are four main kinds of proteolytic enzymes, generally named after the residues at the active site (NOT the places where they cause breakages).

1. Serine proteases- contain serine in the active site
2. Thiol proteases- contain cysteine in the active site
3. Metalloproteases- contain a metal ion in the active site
4. Acid (Aspartate) proteases- operate at acidic pHs, contain two essential aspartate residues in the active site

Serine proteases

Serine proteases contain a "catalytic triad" of three essential residues: serine, histidine and aspartic acid (aspartate). The first steps involve general base catalysis followed by general acid catalysis: the histidine acts as a general base allowing the -OH group on serine to nucleophilically attack the carbonyl carbon. After the breakage of the C-N bond, the carbonyl group is still attached to the serine, whereas the amino group is released from histidine (histidine donates an H, like an acid). This allows histidine to then act as a general base again, stealing an H from water to allow the -OH to attack the carbonyl carbon and release the carboxyl group from serine. (The H that histidine stole gets transferred back to serine- again, kinda like an acid.)

As for the aspartate group? That doesn't directly contribute, but its negative charge at biological pH may help stabilise histidine as it becomes more positively charged during parts of the process.

There are three main serine proteases that you should know: trypsin, chymotrypsin and elastase. Trypsin breaks bonds after lysine and arginine. Chymotrypsin breaks bonds after large, bulky amino acids like tryptophan and tyrosine whereas elastase breaks bonds after little things like alanine and valine. (This is by no means a comprehensive list.)

Thiol proteases

Thiol proteases act in a similar way to serine proteases, but instead of a catalytic triad, they have a "catalytic diad." This diad includes histidine and cysteine. Thiol proteases such as papain have a broad specificity. They do have some preferences, but they have so many preferences that they might as well not have preferences- kinda like people who prefer to drink on all days ending in Y.

Metalloproteases

Metalloproteases seem to act mainly by metal ion catalysis: the water nucleophilically attacks the carbonyl carbon, helped out by the metal ion, which helps to stabilise the negatively-charged carbonyl oxygen in the transition state. Glutamate, which deprotonates water, helps out too. A tyrosine residue may also play a role in breaking the bond, but this is unclear.

Metalloproteases tend not to have specificity for any particular amino acid- they tend to simply hydrolyse the last bond in the chain.

Acid (Aspartate) proteases

As their name suggests, acid proteases work best at acidic pHs. They have two essential aspartate residues. One of these is usually deprotonated, while the other is protonated. The deprotonated aspartate residue acts as a general base to help water attack, whereas the protonated aspartate residue acts as a general acid to make the carbonyl carbon more positively charged, thus helping along the process. This means that the originally protonated aspartate is now deprotonated, and vice versa. The aspartate residues are close enough for this to reverse, and then more general base/general acid action can occur to allow the bond to break.

Acid proteases tend to catalyse bonds between aromatic amino acids, such as phenylalanine and tyrosine.

One example of an acid protease that you should know is HIV Protease. I've written about it as well as drugs that target it in a previous post for PHAR2210.