Ampicillin – to some bacteria it’s a villain – this irreversible enzyme inhibitor causes bacteria trouble when it prevents them from preventing the popping of their cell membrane bubble. But stick in an Amp resistance gene and bacterial colonies will be seen – Amp won’t cause them any trouble – so they’ll continue to double and double. The resistance gene will protect, and thus we can select, cells that’ve taken in a plasmid with our gene of interest within! 

I hate to NAG but did you remember to add AMPICILLIN to weed out cells that *lact* the BETA-LACTAMASE gene? Β-lactamase provides RESISTANCE to the antibiotic AMPICILLIN (Amp) by “popping” Amp’s LACTAM ring before Amp can “pop” the bacteria’s cell wall. And how it does it is *amase*-ing.

Bacteria shield themselves by building cell walls to reinforce their fatty cell membranes.  In addition to keeping stuff *out* the wall’s important for keeping stuff *in.* It’s kinda like a water balloon 🎈 if you let too much water in, the pressure of the water pushing against the balloon skin gets too high & the balloon will pop. If this happens in cells we call it LYSIS

In MOLECULAR CLONING we stick a gene we’re interested in into a circular piece of DNA called a plasmid ⭕️ then stick that into host cells (often harmless bacteria) to make more copies of that gene &/or the protein it codes for. LATER we’ll *want* to lyse these cells to get our protein out. But, while they’re still growing, we want to keep all the protein & DNA-making machinery in the cells so the cells can make the DNA & protein for us.

BUT we only want the “right” cells to stay intact – those that actually have our plasmid. We can SELECT for just those cells by putting an ANTIBIOTIC RESISTANCE GENE into our plasmid alongside our gene, then spiking the bacteria food w/the corresponding antibiotic so that only cells with the plasmid can survive & grow. More here:

For example, we can design a plasmid to hold our gene & the bla gene, which has the instructions for making Β-lactamase, with provides resistance to ampicillin (Amp).

Amp is a BETA-LACTAM antibiotic. It gets this classification because its chemical structure contains something called a B-lactam. A lactam has nitrogen (N) in a 4-sided ring (so basically a square…) N likes being in *some* rings. But it’s less happy to be in other rings. Its relative happiness depends in part on the BOND ANGLES & the potential for RESONANCE.

The N in imidazole rings (like those in Histidine’s (one of the AMINO ACID protein building blocks) side chain) are happy to be there because they can contribute to (and benefit from) RESONANCE STABILIZATION (electron delocalization). Basically this is a kind-of communal sharing of extra electrons (e⁻) that makes all the sharers happy & a 6-sided ring makes for great bond angles. 5-sided rings work too, but 4 (like you have in Amp) is pushing it… The “corners” have to be “sharper” than the bonds would like so you get RING STRAIN

And, to make things worse, Amp’s 4-sided B-lactam ring is fused to another, 5-sided thiazolidine ring. This puts the N in a physically awkward position where to get them to join at all you have to make them “pucker.” This pushes the N out of line with the neighboring C=O, so it can’t resonate with it like it would in a “normal” AMIDE (an N next to a carbonyl group (C=O)). So you don’t get stabilization from e⁻ delocalization.

You can find “normal” amides in a protein’s backbone – and we give them a special name –  we call the amide bonds between the amino acid building blocks of proteins PEPTIDE BONDS & they’re planar – the N, C, & O are in line. This allows for resonance (that communal e⁻ sharing). In addition to physically stiffening the protein backbone, the amides chemically protect the protein chain because the resonance means that *these* amides are NOT very reactive, which is good or else our proteins would get broken apart easily! 

BUT the non-planarity of the “weird” amide in a Β-lactam prevents this resonance. Combine this w/ring strain & the amide in the LACTAM *is* reactive. Why does all this matter? It gives bacteria a way to build cell walls, and it also gives us an “Achille’s heel” to target those bacteria w/antibiotics!

Bacteria build their cell walls by linking sugar chains w/amino acid linkers. If you put a lot of amino acids together you get a protein. But you can also link together just a few to get a peptide. And you can link them to sugars to get PEPTIDOGLYCANS. It’s like an expansion pack for your sugar LEGO set. It gives you more options

Bacteria use these PEPTIDOGLYCANS (aka MUREIN) to build their cell walls. Its sugar (glycan) part’s a POLYSACCHARIDE (long chain of sugars) made up of alternating N-acetylglucosamine (NAG aka GlcNAc) & N-acetylmuramic acid (NAM aka MurNAC) & the peptide part’s a short peptide (typically 4-5 amino acids long, sometimes with a “branch”). Different types of bacteria can have different peptide sequences, but they share the same NAG-NAM-NAM-NAM sugar part. The peptides can bind each other to “crosslink” the sugar strands & make a strong multi-layer cell wall. BUT they need help doing this…

A TRANSPEPTIDASE enzyme coordinates this linking. It’s an ENZYME meaning that it gets *used* but NOT *used up.* Enzymes coordinate reactions & might get temporarily modified in the process, BUT then when it’s finished it’s back to its GO state 👉 any changes that were made are “erased”

The transpeptidase has a hydroxyl (-OH) group sticking out from a Serine (Ser) amino acid in its active site (the place where the action occurs). This acts as a NUCLEOPHILE (something that has “extra” e⁻ it wants to share) to latch onto one chain’s peptide. This “holds it still” so that the end of another peptide from a neighboring chain can grab it. This works like a “pass-off” – in the middle of the process, the enzyme *has* been modified (it’s bound to the 1st peptide). BUT it then hands that peptide over to the 2nd & is freed again. More on such nucleophilic substitution reactions here:

Ampicillin looks like the ends of the peptides, so it “tricks” the transpeptidase into binding it similarly to how the peptide would bind. BUT when Amp binds the transpeptidase, it DOES NOT get “passed off” 👉 instead it gets stuck. It forms a “permanent” bond w/it so it can’t get back to “GO” & can’t do its job of strengthening the cell wall. So the balloon weakens & pops (cell lyses) killing the cell

Ampicillin is thus an enzyme inhibitor. But, unlike the one’s we looked at yesterday  (competitive, noncompetitive, & uncompetitive)

The types of enzyme inhibitors we looked at before were “reversible” inhibitors – they only bound the enzyme through weak, non-covalent bonds – I call them weak and, individually they are (because their electrons are just hanging out near each other, not actually shacking up (no electron sharing)) – but a lot of weak interactions can lead to a pretty strong binding.

But each of those individual interactions is vulnerable, so it can kinda “peel off” if something better comes along. So reversible inhibitors can be “diluted out” so that if the inhibitor falls off, the enzyme is unlikely to easily find another inhibitor to bind to.

But here, with penicillin, we have an IRREVERSIBLE INHIBITOR – it can’t fall off – so even if you remove all the excess inhibitor so that if it were to fall out there wouldn’t be more to bind, it wouldn’t matter because it can’t fall off!

Penicillin is an example of a special type of irreversible inhibition that’s aka “suicide inhibition” (aka “suicide inactivation” aka “mechanism-based inhibition”). In these cases, the irreversible complex is formed during the course of the normal catalysis reaction – it gets partway there and then gets stuck. These “suicide inhibitors” are similar to the competitive inhibitors in that they bind the active site and prevent the “real deal” from binding, but unlike competitive inhibitors you can’t outcompete them. 

Let’s take a closer look at what’s happening at the biochemical level. Because human cells don’t have cell walls (just a fatty cell membrane) cell walls are a great target for antibiotics since we don’t have to worry about them disrupting our cell walls since we don’t have any to disrupt! Another way Amp’s attack is bacteria-specific takes advantage of the bacteria-specific nature of those peptide ends.

STEREOCHEMISTRY refers to the relative 3-D orientation of bonded atoms. Amino acids can be in the “L” form or the “D” form 👉 the same atoms are bonded together, but their 3-D orientation or “handedness” is different. More here:

Normally (in proteins) amino acids are in the “L” form. BUT the end amino acids in these peptide linkers are a pair of “D” alanines (D-Ala-D-Ala). This protects the bacteria from normal protease enzymes (protein chewers) because, like a right hand & a left-handed baseball glove, they don’t fit the proteases’ active sites. BUT the D-Ala-D-Ala *does* fit the active site of the transpeptidase. AND so does Amp because it mimics this “unusual” amino acid pair

Transpeptidase attacks the 1st of these D-Ala & kicks out the last one as a “leaving group.” But when it attacks Amp, the leaving group can’t leave – it’s physically tethered there because cutting a ring once only “un-ring-ifies” it – you still have a chain. And it’s not going anywhere – you’ve irreversibly inhibited the enzyme.

Cells w/the bla-containing plasmid are protected from this because Β-lactamase inactivates Amp by attacking its Β-lactam ring & popping it open. In covalent bonds like those linking the atoms in the ring, neighboring atoms bond by sharing electrons (e⁻). On its own, neither atom has as many e⁻ as it wants so they share. BUT because the ring’s under strain (kinda like a compressed spring), it’s eager to pop open if there’s something to give it the e⁻ it needs. The C of the carbonyl (C=O) is ELECTROPHILIC (it *wants* e⁻). This is a perfect match for the NUCLEOPHILIC active site residues of Β-lactamase. It attacks ⏩ the ring breaks open ⏩ no longer can bind the transpeptidase ⏩ transpeptidase goes about its business as usual.

This TRANSPEPTIDASE is aka PENICILLIN BINDING PROTEIN (PBP) because, just like it binds Amp, it binds penicillin. Amp is actually a modified version of penicillin. Penicillin can be modified in different ways to change its properties – just make sure you keep that beta-lactam part intact. By changing the other chemical groups you can optimize antibiotics to work better on different types of bacteria and/or avoid B-lactamase inhibition. Other penicillin derivatives include methicillin, oxacillin, amoxicillin, & ticarcillin. And other Β-lactam antibiotics include cephalosporins (Ceph), monobactams, carbapenems

Note: A problem with bla/amp as opposed to other antibiotic resistance gene/antibiotic pairs is that B-lactamase gets SECRETED 👉 the cells ship some out to destroy Amp in their food before it even gets to them 👉 in addition to protecting itself, this protects the cells around it, some of which might not have your plasmid 👉 can lead to growth of plasmid-less cells 😬 if you’re growing on a plate these will show up as satellite colonies (clusters of colonies popping up around the good colonies). more here:

You might have heard of “Gram negative” & “Gram positive” bacteria… “Gram” is a type of stain discovered by a guy named Gram & it takes advantage of peptidoglycans to help classify bacteria. Bacteria that have strong, thick, cell walls (coming from up to 40 layers of peptidoglycan) retain the stain so show up dark purple & are “Gram POSITIVE.” An example is Staph aureus. Gram NEGATIVE bacteria have weaker cell walls with only a couple peptidoglycan layers (but they have other sources of additional protection including an “outer membrane”) so the stain leaks out and the cells appear lighter. An example is E. coli. Penicillin works better on Gram positive bacteria which depend more on peptidoglycan & don’t have an outer membrane making it harder for the drug to get to where that peptidoglycan’s at.

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉

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