Penicillin is an antibiotic that kills bacteria by inhibiting an enzyme that crosslinks the bacterial cell wall. Without a strong, crosslinked cell wall, the bacteria explode as a result of the osmotic pressure that exists inside the cell.
By deploying β-lactamase enzymes on it's surface, bacteria can intercept and destroy penicillin before it has a chance to inhibit the crosslinking enzyme. This is one way in which bacteria can evolve resistance to an antibiotic.
Penicillin is known as a beta-lactam antibiotic. It contains an unusual 4-atom lactam ring. β-lactamase opens this ring….and in so doing, inactivates the antibiotic.
This Guided Exploration is based on the structure of β-lactamase as described in the PDB file 5KMW.
In 2016, Patricia Langan and colleagues solved the 3D structure of the Toho 1 β-lactamase with penicillin bound in it's active site. (see figure at top of page). This structure was notable in that it was the first structure to capture the penicillin in the enzyme's active site.
QUESTION: Why were Langan and colleagues successful in capturing penicillin in the active site... while many other groups had failed? This Guided Exploration will address this question.
Click Here to look at the structure of B-lactamase
Click and drag the Jmol image to the right to spin it around to examine it's secondary structure.
QUESTION: Do you see a 5-stranded beta-sheet and clusters of alpha helices ... all joined by short loops of protein structure?
Click Here to show the secondary structures color-coded (yellow beta-sheet and red alpha helices)
Now let's find the active site. When penicillin binds to beta-lactamase, it forms a covalent bond to Ser70. So,... where is Ser70?
Click Here to show a Jmol image with the sidechain of Ser70 displayed. Notice that the active site appears to be located in a groove between two compact domains of the enzyme.
Now let's display the penicillin bound in the active site of the enzyme.
Click Here to display the penicillin.
Zoom in on Ser70 -- and notice that the penicillin is covalently bound to this amino acid. Notice also that the 4-atom lactam ring of the penicillin has been opened -- inactivating the penicillin.
Now back to our original question -- Why was Langan et.al. successful in trapping penicillin in the active site of beta-lactamase, while many other research groups had failed to do this?
The answer to this question is found in amino acid 166. Langan and colleagues knew from other research into the chemistry of this active site that Glu166 played a critical role in destabilizing the covalent bond that connected penicillin to the Ser70 sidechain. Normally this covalent bond only lasts for a very short time before it is cleaved by Glu166, releasing the penicillin.
Find amino acid #166 in the Jmol image to the right. (If you 'hover' your mouse over each amino acid -- its identity will be reported on the screen.) When you find amino acid 166 you will see that it is not Glu (Glutamic acid). Instead, it is Ala (Alanine).
So,... the answer to the above question is that Langan et.al. created a variant form of beta-lactamase in which Glu166 was changed to Ala166. As a result, the covalent bond connecting penicillin to the enzyme was not broken....and they observed the antibiotic bound in the active site.
If you zoom in on the penicillin, you will notice that it is now covalently boned to the sidechain of Ser70.
Click Here to see a zoomed in view of the active site -- with the Ala166 sidechain displayed as a green sphere.
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