BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

PART V. MOLECULAR PHYSIOLOGY

CHAPTER 32. BACTERIAL CELL WALLS

32.9. Penicillin Causes the Death of Growing Bacteria by Inhibiting Cell Wall Synthesis

Penicillin was discovered quite serendipitously in 1928 by Alexander Fleming.

While working with various strains of staphylococci, some culture plates were set aside in a laboratory collection and examined from time to time. Because the plates had to be opened when examined, various airborne microorganisms inevitably contaminated them. It was noticed that around a large colony of a contaminating mold, the staphylococcal colonies became transparent and had clearly undergone lysis.

The mold was subcultured, and preliminary studies were initiated on the bacteriological substance that was evidently synthesized by the mold and diffused into the surrounding medium. It was found that broth in which the mold had been cultured for 1–2 weeks at room Temperature acquired marked inhibitory, bactericidal, and bacteriolytic properties against many common pathogenic Bacteria.

It was further discovered that an extract of the *Penicillium* mold produced no noticeable toxic effects when administered to animals. This served as an incentive for further research. However, when Fleming attempted to concentrate and purify the active antibiotic, he noted: "Penicillin is very labile, and despite all our efforts we failed to get it concentrated. We were bacteriologists, not chemists, and our relatively simple Procedures did not meet with success."

Ten years passed before penicillin was revisited. Pathologist Howard Florey and biochemist Ernst Chain carried out a series of in-depth studies that culminated in the isolation, chemical characterization, and clinical use of this antibiotic. Penicillin consists of a thiazolidine ring fused with a β-lactam ring, with one of various substituents (R) attached at a specific position via a peptide bond. In benzylpenicillin, for example, R is a benzyl group (Figs. 32.13 and 32.14). This Structure is subject to various transformations, which accounts for the lability of penicillin first encountered by Fleming. The β-lactam ring is particularly unstable; as will be shown below, this property is directly related to the antibiotic activity of penicillin.

Class="center">Fig. 32.13. The highly reactive site of penicillin is the peptide bond of the β-lactam ring. Various substituents (variable group) can occupy the R position; in benzylpenicillin, R is a benzyl group

Fig. 32.14. Model of benzylpenicillin

In 1957, Joshua Lederberg demonstrated that bacteria normally sensitive to penicillin can grow in the presence of this antibiotic if a hypertonic medium is used. Microorganisms grown in this manner, known as protoplasts, lack Cell walls and therefore undergo lysis when transferred to a normal medium. From this, it was concluded that penicillin prevents the synthesis of bacterial cell walls. In 1965, James Park and Jack Strominger independently concluded that penicillin blocks The final stage of Biosynthesis, specifically cross-link formation.

32.10. Penicillin Blocks Cell Wall Synthesis by Inhibiting the Transpeptidation Reaction

Penicillin inhibits the transpeptidase that cross-links the proteoglycan chains:

Under normal reaction conditions, transpeptidase forms an acyl intermediate with the penultimate D-Alanine residue of the peptide (Fig. 32.15). This intermediate then reacts with the amino group of the terminal Glycine of another peptide. Recent studies have shown that penicillin inhibits transpeptidase by forming a covalent bond with a Serine residue in the Active Site of the enzyme (Fig. 32.16). The penicilloyl-enzyme complex does not undergo deacylation, making the inhibition of transpeptidase by penicillin irreversible.

Fig. 32.15. During the transpeptidation reaction, an acyl-enzyme intermediate is formed

Fig. 32.16. Formation of an indefinitely stable enzyme complex by penicillin

Why is penicillin such an effective transpeptidase inhibitor? Molecular modeling has revealed that penicillin resembles one of the enzyme's substrates, namely acyl-D-Ala-D-Ala (Fig. 32.17). In addition, the four-membered β-lactam ring of penicillin is in a strained conformation, which renders the peptide bond within it highly reactive. In essence, penicillin appears to mimic the transition-state STRUCTURE OF THE enzyme's normal substrate. In other words, penicillin is a transition-state analog.

Fig. 32.17. The conformation of penicillin in the region of the highly reactive peptide bond (A) resembles the proposed conformation of the R-D-Ala-D-Ala structure (B) in the Transition State during the transpeptidation reaction

It is instructive to compare penicillin with other irreversible Enzyme Inhibitors. The stable, inactive penicillitoyl-enzyme complex is completely analogous to the phosphoryl-enzyme complex formed during the interaction of organofluorophosphates with acetylcholinesterase or Serine proteinases. However, the Specificity of penicillin is much higher than that of fluorophosphate inhibitors. The target of penicillin action is strictly defined by its structural similarity to the terminal D-Ala-D-Ala segment of the newly forming peptidoglycan chain. A consequence of this exceptional specificity is the low toxicity of penicillin, which is so valuable in clinical practice. The Human Body lacks any enzyme that recognizes D-Ala-D-Ala, and therefore penicillin cannot interfere with the function of our own Enzymes.

32.11. Some bacteria are resistant to penicillin because they synthesize an enzyme that destroys it

A number of bacteria synthesize penicillinase, an enzyme capable of cleaving the amide bond in the β-lactam ring of penicillin to yield penicilloic acid, which lacks antibiotic activity:

A series of structurally related penicillinases have been isolated, with a molecular mass of about 30 kDa and a high turnover number (on the order of 10³ s^-1). Enzyme activity depends largely on The Nature of the R group attached to the β-lactam ring of penicillin. Therefore, Semisynthetic Penicillins with R groups that protect them from penicillinase action are of great clinical value.

The Gene encoding penicillinase is located on various Plasmids (Section 31.6). In some bacterial species, these extrachromosomal genetic elements can rapidly appear and disappear. In a number of bacterial strains, penicillinase is an inducible enzyme. Apparently, penicillinase evolved as a detoxification mechanism, since it is characteristic only of microorganisms whose Cell wall is composed of peptidoglycan. Judging by the fact that the loss of the penicillinase gene causes no cell damage in the absence of the antibiotic, the enzyme likely has no other function besides the inactivation of penicillin. Furthermore, There is a strong correlation between the degree of resistance to penicillin and overall penicillinase activity.