Penicillin: Structures and Functions

Corey Atteridge and Mark Tromblay

CH 0337 J-Term Course

Middlebury College

Index

• Introduction/Topic

• Structural Features of Penicillin, Transpeptidase, and Penicillinase

• How this Web Page was Made/References

Introduction/Topic

Penicillin G was first discovered by the French Medical student Ernest Duchesne in 1896. Due to its instability, chemists of the time were unable to produce the drug for any use. As a result, penicillin was not extensively researched until Alexander Fleming began working with penicillin in 1928. Fifteen years later, the drug was mass produced. Penicillin G is derived from the fungus Penicilliumchrysogenum. In nature, the fungus produces this antibiotic when its food resources are limited in order to kill surrounding bacteria. Since that time new Penicillins (Penicillin V, Ampicillin, Methicillin, etc.) have been created or discovered.

Penicillins are characterized by a structural feature known as a Beta-Lactam ring. Other antibiotics like Cephalosporins also share this structural feature. This four membered ring makes the molecule thermodynamically unstable, thus accounting for the trouble that scientists had when working on it in the beginning of this century. This ring is also easily hydrolyzed in the presence of acid, therefore Penicillin derived from natural sources needs to be modified during manufacturing for oral administration, so that it will not be destroyed in the stomach.

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How Penicillin Functions

We chose this project to gain a better understanding of Penicillin and how it works at a molecular and cellular level. The topic of antibiotic resistance was also a primary interest. The movie presentation (see next paragraph) is a representation of Penicillin's deleterious effects on non-resistant bacteria. We wanted to discover why and how this process occurs.

The effectiveness of antibacterial drugs is directly related to the results of a Gram stain. Bacteria can be classified as gram positive or gram negative depending on how well the cell wall retains Gentian violet stain. Gram positive bacteria have thick peptidoglycan cell walls which contain cross-linked polysaccharides. Penicillin is mostly effective against this type of bacteria, since it kills bacteria by disrupting the peptidoglycan cross-linking process. Gram negative bacteria, including E. Coli , have thin polysaccharide walls overlaid by thin layers of lipopolysaccharides. The protective barrier in Gram negative bacteria prevents Gentian violet stain from reaching the peptidoglycan target. This indicates that it is harder for Penicillin to penetrate the lipopolysaccharide layer. Some Penicillin must be able to get through, as is demonstrated by the fact that it kills Gram negative bacteria although not as efficiently as Gram positive. The structure of bacterial cell walls can be found here. Penicillin functions by preventing proper cross-linking of the peptidoglycan layer of growing cells. For a movie of the effects of Penicillin on growing E.Coli cells click here. It is important to note that peptidoglycan only needs to be cross linked when the bacteria are creating new cell walls structures; during periods of growth or division. Penicillin has no effect on peptidoglycan strands which are already cross linked.

Peptidoglycan is a protective mesh made up of long chains of two alternating sugars, N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG). When the formation of this mesh is disrupted by Penicillin the mesh is no longer protective and the cell is destroyed. Since the peptidoglycan is the ultimate target of Penicillin, eukaryotic cells are not affected by the drug since they do not contain peptidoglycan.

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Structural Features of Penicillin, Transpeptidase, and Penicillinase

This is an image of D-alanyl-D-alanine Carboxypeptidase (a transpeptidase) bound to a Penicillin analog. The protein is colored from the N-terminus (dark blue) to the C-terminus (green). This Carboxypeptidase, also known as a Penicillin Binding Protein (PBP), attaches to D-alanine residues from the carboxy termini of amino acid side-chains of two NAM sugars on adjacent strands of peptidoglycan and subsequently joins the two strands together, cross-linking the peptidoglycan's NAM sugars with a pentaglycine bridge. To see an enlarged image of this enzyme's active site, click on the D-alanyl-D-alanine Carboxypeptidase image.

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Penicillin inhibits the Carboxypeptidase's crosslinking capabilities by acylating the enzyme and binding irreversibly to it's catalytic site. In order to see the structural resemblance between D-alanyl-D-alanine and penicillin, click on this image. Bacteria which produce Penicillin naturally have altered PBP's which do not bind to Penicillin as easily, if at all; and as a result are resistant to its effects.

Penicillin has become a widely used antibiotic against a variety of bacterial organisms. However, some bacteria have developed resistance to the drug. Spontaneous mutations in bacterial chromosomes result in changes in receptor sites and prevent penicillin from binding to the PBP's. Therefore, penicillin cannot inhibit transpeptidation of the growing bacterial cell wall. This is an image of Beta-Lactamase, also known as Penicillinase. To see an enlarged image of the enzyme's active site, click on the Penicillinase image. A few reasons why bacterial resistance to antibiotics has become such a problem are as follows. One is that penicillin, and other antibiotics may be over-prescribed: if people take penicillin when they have just a minor bacterial infection that their immune systems may have been able to combat, then they are merely increasing the chances that a resistant mutant will emerge. By destroying the non-resistant bacteria, the resistant forms will proliferate due to lack of competition. This logic can also be applied to people who do need to take antibiotic but do not take enough of a dose, or enough dosages.

Penicillinase deactivates Penicillin by hydrolyzing its four membered Beta lactam ring. This spacefilling image of Penicillin shows where and how this hydrolysis occurs. New Penicillins have been created which are somewhat Penicillinase resistant, but it probably only a matter of time before a strain of bacteria will arise that makes a different type of Penicillinase which effectively deactivates these new Penicillins.

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The Future

Bacterial resistance to Penicillin comes in many forms. A mutation in bacterial DNA can limit the permeability of the cell wall, cause the host to make proteins which produce enzymes to inactivate the antibacterial agent (Penicillinases), and alter cross-linking enzymes so that Penicillin can no longer bind to them. These mutations can be gradual mutations in the bacterial genes or they can be acquired from whole genes from another bacteria.

Antibiotic resistance is a serious issue in drug therapy. A report from the New England Journal of Medicine (NEJM) explained how researchers have identified bacteria in patients that are resistant to all available forms of antibiotic treatments. One example of a bacteria that has demonstrated considerable resistance to antibiotic treatment includes Streptococcus pneumoniae. Another report from NEJM identified 6.6 percent of pneumococcus strains as being completely resistant. Clearly, researchers must meet this challenge by producing antibacterial agents that will overcome the defenses within these resistant strains. But as more antibiotics have come into existence, more strains of bacteria have as well.

Some possible solutions to this problem are: 1) To educate others not to take antibiotics merely if they have some left over from their last prescription and think they may be coming down with something (there are people who do this!) 2) To educate others that they should use the full prescription in the first place; to kill those bacteria and kill them completely (if they aren't already resistant, they may develop resistance if you just wound them).