How Proteases and Protease Inhibitors Work

Proteases: wild, mysterious, destructive.  What are these untamed elements ravaging your precious lysate?

How can a drop of EDTA or a smidge of “cocktail” protect that sample, which is gently cradling your hopes, your dreams, and your desire to survive the next lab meeting?

Brace yourself for a biochem flashback: in this article, we’ll explain the what and how of proteases and protease inhibitors, and we’ll do it with phrases like “reversible competitive inhibition” and “zymogen.”

Proteases: the good, the bad, the hydrolytic

All organisms have proteolytic and phosphorylating enzymes.  You.  Me.  Helicoverpa armigera.  While involved in everything from host defense to wound healing to viral replication, proteases can be broadly lumped into two camps based on what’s in their active site and how it interacts with the target peptide bond.  Camp One hydrolyzes the bond through nucleophilic activation of a serine, threonine, or cysteine at its active site.  Camp Two, which includes the aspartic, glutamic, and metalloproteases, prefers activating water itself for the hydrolysis.

These tidy camps break down under questioning about preferred targets and substrate affinity.  Although Camp One’s serine proteases all agree that an active site serine is superior to the plebeian cysteine, they still have tense family dinners because of each protease’s different target preferences and structure.  These structural differences drive a protease’s specificity for where it will attack: terminal hydrolysis for the exopeptidases, internal hydrolysis for the endopeptidases.  Although the exopeptidases only choose between the amino or carboxyl terminus, the endopeptidases are gloriously picky.  To belabor the serine example, our friend trypsin hydrolyzes on the carboxyl side of positively charged arginine or lysine, unless these are followed by a proline.  And while its buddy chymotrypsin is also a serine protease, chymotrypsin simply doesn’t touch arginine or lysine and really just prefers aromatics like tyrosine, tryptophan, or phenylalanine.

The last thing a cell wants is these enzymes running amok, rampaging willy-nilly through the cytoplasm, chewing off-target, and generally behaving like – I say this with great familiarity – Ohio State football fans.  Regulation and segregation are the cell’s prime approaches for dealing with these enzymes: regulation by, for example, holding them in an inactive precursor state (a “zymogen”) and segregation to the lysosome or secretory pathways, among other locations.

Inhibitors: You can’t stop with just one.

All that chemical lysis, scraping, sonication, and freeze-thawing you inflict throws the whole system into chaos.  No peptide bond is safe!  Exopeptidases attack the ends of amino acid chains while endopeptidases hydrolyze their internal links.  Kinases phosphorylate and dephosphorylate at will.  Good luck quantifying NOS induction or making a case for p53 activation by Western.  Without inhibitors, your results will be inconsistent, your lab meetings agonizing, and your graduation date a pipe dream.

Tragically, there is no Wunderkind inhibitor that takes out all proteases, halts every phosphorylation event, and does some light tidying-up about the centrifuges.  It takes a “cocktail” of approaches to stave off proteolysis.  Protease inhibitors can act irreversibly, reversibly, and reversibly-under-duress, and can be anything from small molecules like sodium fluoride to big honkin’ polypeptide analogues of evolution’s own microbial inhibitors.  They can compete with substrates for an active site – sometimes destroying the site in the process – or selfishly hoard the precious, precious cations needed for protease function.

Competitive inhibitors act by binding in a substrate-like manner to the active site to block it or by binding and destroying it through modifications such as esterification.  Many large competitive inhibitors boost their affinity and specificity by binding to the overlooked wallflower regions neighboring the hogs-all-the-glory active site.  You have likely already come across competitive inhibitors.  They include all sorts, from aprotinin, a 6 kDa polypeptide serine protease that is reversible if you ask politely at pH extremes, to sodium orthovanadate, a small molecule which inhibits the ever-gullible protein phosphotyrosyl phosphatases.

By contrast, the toddler-like chelators (motto: MINE!) take advantage of their enhanced affinity for metal ions to complex with them so that nobody else can react with them.  This matters because metalloproteases need zinc, magnesium, manganese, calcium, or even cobalt in their active site to activate water to hydrolyze peptide bonds to meet their quarterly hydrolysis goals.  Take away that zinc with EDTA, complex up that calcium with EGTA, and it’s bye-bye metalloprotease activity.

Conclusion: To defeat the protease, you must become the protease.

Proteases are just some of the destructive forces nature aims at your beloved cell lysates, and protease inhibitors are one of the ways that the clever scientist can, judo-like, turn the universe against itself.  For more information on beating the universe into submission, check out Emily Crow’s “How To Preserve Your Samples In Western Blotting”, Jode Plank’s “Streamline Your Western Blots”, and these guides to protease inhibition from Abcam and Pierce.

References:

Abcam.  (2012) Western blotting – a detailed guide.

Farady, C., and Craik, C.  (2010) Mechanisms of macromolecular protease inhibitors.  Chembiochem 11(17): 2341-2346.

Hooper, N.  (2002) Proteases: a primer.  Essays in Biochemistry 38: 1-8.

Rawlings, N., et al.  (2012) MEROPS: the database of proeolytic enzymes, their substrates and inhibitors.  Nucleic Acids Res 40: D343-350.  Database available here.

Thermo Fisher Scientific.  (2012)  Protease and Phosphatase Inhibitors.