Tuesday, June 12, 2007

How Penicillin Works to Kill Bacteria

 
Bacterial cell walls are made of peptidoglycan [Bacteria Have Cell Walls]. In order to form a rigid structure, the polysaccharide chains (glycans) are linked together by peptide crosslinks. The first step in the formation of the crosslinks involves attachment of a short five residue peptide to the MurNAc sugar in the polysaccharide. This peptide ends in two D-Alanine (D-Ala) residues.

This peptide is further modified by attaching an additional peptide to the middle of the first one creating a branched structure. Finally the peptide of one of the polysaccharide molecules is attached to another to form the crosslink. The reaction is a transpeptidase reaction.

The mechanism is shown in the figure (Lee et al. 2003). The top structure (1) is a polysaccharideMurNAc-GlcNAc) with the first peptide already bound. Note that it ends in two D-Ala residues. The first step in the transpeptidase reaction involves binding of the enzyme (Enzyme-OH) to the D-Ala-D-Ala end of the chain. A reaction takes place in which one of the D-Alanine residues is released and the enzyme become attached to the end of the peptide (2).

In the second step, an adjacent peptidoglycan (purple) (3) with a branched structure is covalently linked to the first peptidoglycan forming a crosslink between the two polysaccharides.

Almost all bacteria have cell walls and they have transpeptidase enzymes that catalyze this reaction. The activity of this enzyme is inhibited by penicillins or β-lactams. Mondays Molecule #30 was Penicillin G, one of many different types of β-lactam that block cell wall formation and kill bacteria.

The mechanism of inhibition is well known. The β-lactam region of the drug resembles the D-Ala-D-Ala end of the peptide to which the transpeptidase enzyme binds. The structures are shown below.


A typical penicillin is shown at the top of the figure. The business part of the molecule is the β-lactam moiety and the "R" represents various groups that can be bound to create different penicillin drugs. The structure of D-Ala-D-Ala is shown below.

The structures of several different transpeptidases have been solved. The enzymes are usually called penicillin-binding proteins or PBP's. Most bacteria have several related versions of PDB genes but all of the enzymes are inhibited by β-lactams.

The figure shows the structure of penicillin-binding protein 1a (PBP1a) from Streptococcus pneumoniae with the bound drug in gray in the grove in the lower right corner of the enzyme (Contreras-Martel et al. 2006). This form of the enzyme is inactive because the drug binds very tightly to the active site and blocks the reaction. That's how penicillin works.


Contreras-Martel, C., Job, V., Di Guilmi, A.M., Vernet, T., Dideberg, O. and Dessen, A. (2006) Crystal structure of penicillin-binding protein 1a (PBP1a) reveals a mutational hotspot implicated in beta-lactam resistance in Streptococcus pneumoniae. J. Mol. Biol. 355:684-96.


Lee, M., Hesek, D., Suvorov, M., Lee,W., Vakulenko, S. and Mobashery, S. (2003) A mechanism-based inhibitor targeting the DD-transpeptidase activity of bacterial penicillin-binding proteins. J. Am. Chem. Soc. 125:16322-16326.

15 comments :

  1. Great post. I didn't know where or how exactly penicillin interfered with bacteria. I just knew they disrupted the cell wall, but not on a molecular level.

    Another day, another lesson.

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  2. Nice. But I still want to know: does Penicillin actually kill individual bacterial cells, by preventing formation and maintenance of cell walls, or does it not actually kill individual cells but instead prevents reproduction by preventing the necessary increase in cell wall structure prior to cell division? In other words, does a bacterial cell in the presence of Penicillin die, or stop growing (and then get picked off by the immune system or whatever)?

    I'm betting that Penicillin resistance will appear in a future post.

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  3. Penicillin kills by disrupting the crosslinking reaction. In dividing cells, a balance of degradation and synthesis reactions are crucial to the proper septum formation and division. By disrupting the formation of new crosslinks, the penicillin causes an autolytic destruction of the cell by cell wall turnover enzymes. This can actually be used in the lab as a positive selection for auxotrophs, by adding penicillin to a defined medium for growing your (penicillin sensitive bug of interest). When it grows, penicillin kills it. Therefore the auxotrophs (mutants that can't grow because a required nutrient is lacking) survive. I always wanted to try that one...

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  4. Thanks Larry,

    Another satisfying day at Sandwalk.

    In my biochemistry and pharmacology days, the mechanisms of action(MOA) for many common pharmaceutical agents were still in the "working on it" file.

    I'd be interested in what you have to say about how the elucidations of active sites, MOA's relates to the proliferation of "designer drugs." That might be a bit much for a comment response, but, if it interests you, maybe it could be a future post topic.

    Thanks again.

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  5. By disrupting the formation of new crosslinks, the penicillin causes an autolytic destruction of the cell by cell wall turnover enzymes.

    Thanks, Paul. I was looking for the link between "chemical reaction does not happen" and "cell destroyed" - apparently, that link is in the form of other cell wall maintenance enzymes that are not down-regulated in the presence of Penicillin... which suggests one possible mode of resistance.

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  6. I can't figure out how penicillin mimics D-Ala-D-Ala. In the structure of penicillin, I can see one of the D-alanines, but the other one looks like L-alanine to me. If you look at the the Wikipedia image of penicillin, it looks like the beta-lactam ring is in the plane of the page and the NHCOR on the upper left corner of the ring is coming out of the plane of the page. I assume that the "side chain" of this "alanine" is the lactam ring ring carbon that's attached to the S. This means that the configuration about this "alpha carbon" is L, not D. Or am I mistaken?

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  7. other sources seem to look like the wikipedia version as well... maybe it's just that it doesn't mimic the chirality 100%? but then what's the point of saying penicillin mimics D-Alaa-D-Ala...?

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  8. Thank to your kind post, finally I can understand how penicillin work. I'm senior of University studying for chemical engineering in south Korea, and this is my first time to study biological stuffs. Maybe I would like to visit too much here ^^ Thank you again and have a nice day

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  9. thank you! this has helped very much on my science homework :)

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  10. Wow! I am a student, and this really helped me... I am studying on Sir Alexander Fleming. And this was the website that finished it up!!! I really like this site...

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  11. Penicillin-type antibiotics work by:
    A) causing bacterial cells to lyse because their cell membranes become weakened
    B) inhibiting the addition of peptides to NAM in peptidoglycan synthesis
    C) acting as a peptide mimic and inhibiting peptidoglycan synthesis
    D) acting as a NAM mimic and inhibiting peptidoglycan synthesis
    E) inhibiting the addition of new NAG and NAM molecules to peptidoglycan

    I think it can be either "A" or "C". but, I can't figure it out

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  12. Thanks. That was a great explanation. But How do 'broad spectrum penicillins (amoxicillin; methicillin) work, since gram neg bacteria don't have "cell walls" per se? Are there other transpeptidases that are used by gram negatives to maintain/form their cell membranes that these other PCNs work against, or do expanded-spectrum PCNs work against totally different enzymatic reactions?
    Thanks,
    Dr. Faustus

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  13. Thanks. That was a great explanation. But How do 'broad spectrum penicillins (amoxicillin; methicillin) work, since gram neg bacteria don't have "cell walls" per se? Are there other transpeptidases that are used by gram negatives to maintain/form their cell membranes that these other PCNs work against, or do expanded-spectrum PCNs work against totally different enzymatic reactions?
    Thanks,
    Dr. Faustus

    ReplyDelete
  14. How is it that the bacteria die, subsequent to treatment with penicillin? Yes, cell wall synthesis is inhibited, so...

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    Replies
    1. AS the bacteria's cell wall is weakened, it can no longer withstand the high osmotic pressure in it, causing it to lyse. Do check out wikipedia too http://en.wikipedia.org/wiki/Penicillin.Check mode of action.

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