JOURNAL OF ENDODONTICS [ VOL 2, NO 5, MAY 1976
How p e n i c i l l i n w o r k s
Edwin L. Smith, DMD, PhD, Miami, Fla
This a r t i c l e d e s c r i b e s h o w p e n i c i l l i n w o r k s a t the c h e m i c a l l e v e l . It s h o u l d p r o v i d e the e n d o d o n t i s t s o m e i n s i q h t into b a c t e r i a l r e s i s t a n c e to a n d b a c t e r i a l s p e c t r a of p e n i c i l l i n s .
Antibiotics can be classified according to the site of action in the bacterial cells. They usually interfere with one of the following processes: (1) fabrication of bacterial cell wall, (2) permeability of the cell membrane, (3) biosynthesis of deoxyribonucleic acid ( D N A ) , (4) protein biosynthesis, or (5) other metabolic processes. Antibiotics that are used by endodontists for oral infections either interfere with bacterial protein synthesis or the fabrication of bacterial cell walls. Examples of antibiotics that interfere with bacterial protein synthesis are erythromycin, tetracyclines, and lincomycin. Chloramphenicol and streptomycin also interfere with protein synthesis, but they are not generally used for dental infections because of their toxicity. The penicillins and cephalosporins are examples of antibiotics that interfere with bacterial cell wall biosynthesis. This article reviews how bacterial cell wall is made, how enzymes work, and the chemical structure of the penicillin molecule itself.
Bacterial Cell W a l l The bacterial cell, like a human cell, has a cell membrane (Fig 1).
Unlike mammalian cells, however, the bacterial cell has a cell wall, a rigid supporting structure outside of the cell membrane, (Fig 1). When .a bacterial cell enters the blood or tissue fluids of a human, enormous osmatic pressures are built up within the cell, ~ and if it were not for the great strength of the cell wall, the bacterial cell membrane would burst, resulting in a quick death for the bacterium. 1 F o r purposes of discussion, the cell
ce
membrane
I !":i'~';
/ /%,,/1',~'
~bAk, outer
[ murein e
portion
Fig 1--Dividing bacterial cell. Enlarged view (box) shows schematically position o/ cell membrane and two components o/ cell wall: murein and outer accessory portion.
Fig 2--Schematic drawing o/ murein component o] cell wall shows carbohydrate chains (black and white circles) cross-linked by amino acid bridges (black bars). This cross-linking gives murein (and thus cell wall) its great strength.
wall can be divided into two parts (Fig 1). The outer part can be called ,the accessory portion; it is responsible for the differential effect of ,the G r a m stain, carries a number of antigens, and is largely responsible for virulence of bacteria2 It also may play a part in determining antibiotic spectra of penicillins. 3,4 The inner part is a highly crossed-linked structure known as murein and is responsible for the strength of the cell wall e (Fig 2). As a bacterial cell begins to divide, the cell wall must be reproduced. The final step in murein fabrication is the cross-linking step. The penicillins prevent the completion of the crosslinking step, resulting in a cell wall too weak to withstand the enormous intracellular pressure generated within the bacterial wall (Fig 3).
A Special Enzyme: Transpeptidase All chemical reactions in human and bacterial cells are catalyzed by
149
JOURNAL OF ENDODONTICS I VOL 2, NO 5, MAY 1976
\_J
\
B
I \4 TE OF ACTION CROSS- LINKING ENZYME & PENICILLIN
I BEFORECROSS II. CROSSLINK LINK COMPLETED compounds known as enzymes. These compounds speed up chemical reactions so that they proceed at a realistic rate at body temperatures. There are millions of different enzymes in each mammalian and bacterial cell. Each enzyme has the unique characteristic of being very specific in the chemical reaction it will catalyze because there is a special place on the enzyme (known as the active site) that will accept molecules of a particular shape. If a molecule has the wrong shape (or structure), the enzyme will simply "ignore" it. On the other hand, an enzyme will "recognize" molecules that have similar shapes or structures as long as the shapes are the correct ones.
The cross-linking step in murein biosynthesis is catalyzed by an enzyme known as transpeptidase. This enzyme recognizes two parts of the bacterial wall and connects them together to form a cross-link (Fig 3, 4). The penicillin molecule prevents the crosslinking step by interfering with the transpeptidase enzyme. It does this
150
Fig 3 - - L a r g e circles represent carbohydrate strands o/ alternating units o / m u r a m i c acid (A) and glucosamine (B). Strands become very long and there are literally millions o/ strands making up bacterial cell wall. From each muramic acid residue there is a small chain o/ live amino acids (depicted by the live diamonds labeled with numbers 1 through 5) and a bridge o/ /ive small circles representing live residues o/ the amino acid glycine (labeled 6 through 10). This "pentagIycine" bridge is present in some species o/ bacteria, but may vary in structure depending on type o/ bacteria. Transpeptidase enzyme catalyzes cross-linking step, connecting a glycine (10) /rom one chain to an alanine (4) in another chain. In process, an alanine residue (5) is lost, supplying energy to drive reaction. This is only one cross-link; /inished bacterial cell wall has millions o/ strands cross-linked in many places in manner shown in diagram. Finished bacterial cell wall is one giant molecule, highly cross-linked and completely surrounding and protecting /ragile cell membrane o/ bacterium.
because part of the penicillin molecule looks like a part of the bacterial cell wall ~-7 (Fig 4).
GLYC I NE
Penicillin Molecule The penicillins exist as a family of molecules. The penicillin molecule can be divided into a "killer" part and a "modifier" part (Fig 5). The killer part is the same for all penicillins and is the part of the molecule responsible for blocking the action of the transpeptidase enzyme, thus resulting in a weakened bacterial cell wall. The modifier part of the molecule differs among the various penicillins and is responsible, in part, for such things as the bacterial spectra of the various penicillins, the rate of excretion and breakdown, solubility in stomach acids, and absorption (Fig 5). The reason that the killer portion of the penicillin molecule inhibits the transpeptidase enzyme (cross-linking enzyme) is that the penicillin molecule looks like a part of the bacterial cell wall that is cross-linked; that is,
4.,. PENICILLIN MOLECULE
Fig 4 - - U p p e r schematic representation shows how two parts o / c e l l wall /it on cross-linking enzyme (transpeptidase) just be/ore a cross-link is made. Lower picture shows how a penicillin molecule /its on cross-linking enzyme to block its action.
JOURNAL OF ENDODONTICS I VOI. 2, NO 5, MAY 1976
the penicillin molecule is a structural analogue for part of the bacterial cell wall 5-7 (Fig 6). It is believed that when a bacterial cell is dividing in the presence of penicillin, the crossqinking enzyme will "mistake" penicillin for ~he bacterial cell wall. The penicillin becomes irreversibly attached to the cross-linking enzyme, thus inactivating it. 5,7 As the bacterial cell divides and makes new bacterial wail, the crosslinking step cannot be completed and a weakened bacterial wall results.
Bacterial Spectra All bacteria thus far studied possess a murein component .in their cell walls. 3 Since the penicillins interfere with murein biosynthesis, why is it that all bacteria are not killed by penicillin; that is, why do the various penicillins have different bacterial spectra? There are two reasons. Some bacteria make an enzyme (penicillinase) that degrades the killer portion of the penicillin molecule 3 (Fig 7). This changes the shape of the penicillin molecule and it no longer is effective in blocking the transpeptidase enzyme. The penicillin is simply destroyed as fast as it can be introduced into the body. Therefore, an effective concentration of penicillin is never reached in the blood and tissues. The second reason is that the bacteria cell wall contains chemical components other than murein. These other components are responsible for the differential effect of the G r a m stain 2 and also prevent certain penicillins from penetrating to the transpeptidase enzyme. ~-5 Thus while penicillin G apparently cannot penetrate the outer portion (accessory portion) of the cell wall of gram-negative bacteria, ampicillin and carbenicillin can. Why? It has been suggested that by changing the modifier portion of the penicillin to a more polar moiety, such as in ampi-
151
JOURNAL OF ENDODONTICS [ VOL 2, NO 5, MAY 1976
cillin and carbenicillin, these molecules more readily penetrate the accessory portion of certain gramnegative bacteria, s
PENICILLIN G
METHI C I LLI N
Good "FIT"of Penicillin G on active site of Penicillinase
Poor "FIT" of Methicillin on active site of Penicillinase
Lack of T o x i c i t y of Penicillin Except in patients who are allergic to it, penicillin has no toxicity. This is because h u m a n cells lack a cell wall and a transpeptidase cross-linking enzyme, and thus penicillin has no action in mammalian cells. For this reason also, penicillin has no ,action on viral cells, which explains why penicillins are of no use in the ~reatment of viral infections such ,as colds and herpes simplex. Viral cells do not have a murein component and have no transpeptidase enzyme; thus there is no place for penicillin to act.
W h y Penicillin is B a c t e r i o c i d a l Bacterial growth in the presence of penicillin is really independent of penicillin. The bacteria will grow and divide and reproduce bacterial cell wall regardless of whether or not the cross-linking step is completed. If the bacterial cell can be maintained in an isotonic solution (that is, where the pressures both inside and outside the bacterial cell are equal), the bacterial cell will survive despite the weakened cell wall. In fac,t, this has been done experimentally; the cells are then called protoplasts. 1 Under normal conditions however, the pressures inside the bacterial cell are greater than outside; and when penicillin is effective, the cell wall and membrane ruptures, the contents of the bacterial cell spill out, and the bacteria cease to exist as living organized structures. P e n i c i U i n s t h a t Resist P e n i c i l l i n a s e Some penicillins such as methicillin are able to resist the action of peni-
152
cillinase, while others such as penicillin G are not. Since penicillinase opens the fl-lactam ring of the killer portion of the penicillin molecule, and both methicillin and penicillin G contain the fl-lactam ring, the question arises as to how methicillin escapes the action of penicillinase. Conceptually, we must speculate that methicillin has a modifier portion that prevents the killer portion of the molecule from fitting the active site of the enzyme properly. Because of this poor "fit" peniciUinase cannot catalyze the splitting of the fl-lactam ring (Fig 8). Summary The mechanism of action of the penicillins at the chemical level has been described. The reason why penicillin is not toxic to humans and viruses, the reason why different penicillins have different bacterial spectra, and bacterial resistance to penicillins also have been explained. Dr. Smith is a second-year endodontic resident at the Veterans Administration Hospital, Miami, Fla 33125. Formerly he was assistant professor of biochemistry and dentistry at .the dental school, University of Alabama in Birmingham. Reprint requests should be directed to Dr. Edwin L. Smith, Veterans Administration Hospital, 1201 NW 16th St, Miami, Fla 33125.
Fig 8---Good fit o/ peni. cillin on penicillinase is necessary for inactivation o] penicillin. Penicillinase-resis}. ant penicillins (such as methi. cillin) may escape action o] penicillinases because shape of modifier portion of penicillin molecule may prevent good fit.
References 1. Sharon, N. The bacterial cell wall. Sci Am 220:92 May 1969. 2. Lehninger, A.L. Sugars, storage polysaccharides, and cell walls, in Biochemistry. The molecular basis of cell structure and function. New York, Worth Publishers Inc., 1970, p 217. 3. Richmond, M.H., and Curtis, N.A.C. The interplay of /3-1actamases and intrinsic factors in the resistance of gram-negative bacteria to penicillins and cephalosporins. Ann NY Acad Sci 235:553 May 1974. 4. Boman, H.G.; Nordstr6m, K.; and Normark, S. Penicillin resistance in Escherichia coli K12: synergism between penicillinases and a barrier in the outer part of the envelope. Ann NY Acad Sci 235:569 May 1974. 5. Strominger, J.L. Enzymatic reactions in bacterial cell wall synthesis sensitive to penicillins, cephalosporins and other antibacterial agents, in Gottlieb, D., and Shaw, P.D. (eds), Antibiotics, Vol 1: Mechanism of action. New York, Springer-Verlag Inc., 1967, p 705. 6. Tipper, D.J., and Strominger, J.L. Mechanism of action of penicillins: a proposal based on their structural similarity to the acyl-D-alanyl-D-alanine. Proc Natl Acad Sci USA 54:1133 Oct 1965. 7. Strominger, J.L.; Willoughby, E.; Kamiryo, T.; BIumberg, P.M.; and Yocum, R.R. Penicillin-sensitive enzymes and penicillin-binding components in bacterial cells. Ann NY Acad Sci 235: 210 May 1974. 8. Butler, K.; English, A.R.; Ray, V.A.; and Timreck, A.E. Carbenicillin: chemistry and mode of action. J Infect Dis 122 (Suppl): S1 Sept 1970.
How p e n i c i l l i n w o r k s
Edwin L. Smith, DMD, PhD, Miami, Fla
This a r t i c l e d e s c r i b e s h o w p e n i c i l l i n w o r k s a t the c h e m i c a l l e v e l . It s h o u l d p r o v i d e the e n d o d o n t i s t s o m e i n s i q h t into b a c t e r i a l r e s i s t a n c e to a n d b a c t e r i a l s p e c t r a of p e n i c i l l i n s .
Antibiotics can be classified according to the site of action in the bacterial cells. They usually interfere with one of the following processes: (1) fabrication of bacterial cell wall, (2) permeability of the cell membrane, (3) biosynthesis of deoxyribonucleic acid ( D N A ) , (4) protein biosynthesis, or (5) other metabolic processes. Antibiotics that are used by endodontists for oral infections either interfere with bacterial protein synthesis or the fabrication of bacterial cell walls. Examples of antibiotics that interfere with bacterial protein synthesis are erythromycin, tetracyclines, and lincomycin. Chloramphenicol and streptomycin also interfere with protein synthesis, but they are not generally used for dental infections because of their toxicity. The penicillins and cephalosporins are examples of antibiotics that interfere with bacterial cell wall biosynthesis. This article reviews how bacterial cell wall is made, how enzymes work, and the chemical structure of the penicillin molecule itself.
Bacterial Cell W a l l The bacterial cell, like a human cell, has a cell membrane (Fig 1).
Unlike mammalian cells, however, the bacterial cell has a cell wall, a rigid supporting structure outside of the cell membrane, (Fig 1). When .a bacterial cell enters the blood or tissue fluids of a human, enormous osmatic pressures are built up within the cell, ~ and if it were not for the great strength of the cell wall, the bacterial cell membrane would burst, resulting in a quick death for the bacterium. 1 F o r purposes of discussion, the cell
ce
membrane
I !":i'~';
/ /%,,/1',~'
~bAk, outer
[ murein e
portion
Fig 1--Dividing bacterial cell. Enlarged view (box) shows schematically position o/ cell membrane and two components o/ cell wall: murein and outer accessory portion.
Fig 2--Schematic drawing o/ murein component o] cell wall shows carbohydrate chains (black and white circles) cross-linked by amino acid bridges (black bars). This cross-linking gives murein (and thus cell wall) its great strength.
wall can be divided into two parts (Fig 1). The outer part can be called ,the accessory portion; it is responsible for the differential effect of ,the G r a m stain, carries a number of antigens, and is largely responsible for virulence of bacteria2 It also may play a part in determining antibiotic spectra of penicillins. 3,4 The inner part is a highly crossed-linked structure known as murein and is responsible for the strength of the cell wall e (Fig 2). As a bacterial cell begins to divide, the cell wall must be reproduced. The final step in murein fabrication is the cross-linking step. The penicillins prevent the completion of the crosslinking step, resulting in a cell wall too weak to withstand the enormous intracellular pressure generated within the bacterial wall (Fig 3).
A Special Enzyme: Transpeptidase All chemical reactions in human and bacterial cells are catalyzed by
149
JOURNAL OF ENDODONTICS I VOL 2, NO 5, MAY 1976
\_J
\
B
I \4 TE OF ACTION CROSS- LINKING ENZYME & PENICILLIN
I BEFORECROSS II. CROSSLINK LINK COMPLETED compounds known as enzymes. These compounds speed up chemical reactions so that they proceed at a realistic rate at body temperatures. There are millions of different enzymes in each mammalian and bacterial cell. Each enzyme has the unique characteristic of being very specific in the chemical reaction it will catalyze because there is a special place on the enzyme (known as the active site) that will accept molecules of a particular shape. If a molecule has the wrong shape (or structure), the enzyme will simply "ignore" it. On the other hand, an enzyme will "recognize" molecules that have similar shapes or structures as long as the shapes are the correct ones.
The cross-linking step in murein biosynthesis is catalyzed by an enzyme known as transpeptidase. This enzyme recognizes two parts of the bacterial wall and connects them together to form a cross-link (Fig 3, 4). The penicillin molecule prevents the crosslinking step by interfering with the transpeptidase enzyme. It does this
150
Fig 3 - - L a r g e circles represent carbohydrate strands o/ alternating units o / m u r a m i c acid (A) and glucosamine (B). Strands become very long and there are literally millions o/ strands making up bacterial cell wall. From each muramic acid residue there is a small chain o/ live amino acids (depicted by the live diamonds labeled with numbers 1 through 5) and a bridge o/ /ive small circles representing live residues o/ the amino acid glycine (labeled 6 through 10). This "pentagIycine" bridge is present in some species o/ bacteria, but may vary in structure depending on type o/ bacteria. Transpeptidase enzyme catalyzes cross-linking step, connecting a glycine (10) /rom one chain to an alanine (4) in another chain. In process, an alanine residue (5) is lost, supplying energy to drive reaction. This is only one cross-link; /inished bacterial cell wall has millions o/ strands cross-linked in many places in manner shown in diagram. Finished bacterial cell wall is one giant molecule, highly cross-linked and completely surrounding and protecting /ragile cell membrane o/ bacterium.
because part of the penicillin molecule looks like a part of the bacterial cell wall ~-7 (Fig 4).
GLYC I NE
Penicillin Molecule The penicillins exist as a family of molecules. The penicillin molecule can be divided into a "killer" part and a "modifier" part (Fig 5). The killer part is the same for all penicillins and is the part of the molecule responsible for blocking the action of the transpeptidase enzyme, thus resulting in a weakened bacterial cell wall. The modifier part of the molecule differs among the various penicillins and is responsible, in part, for such things as the bacterial spectra of the various penicillins, the rate of excretion and breakdown, solubility in stomach acids, and absorption (Fig 5). The reason that the killer portion of the penicillin molecule inhibits the transpeptidase enzyme (cross-linking enzyme) is that the penicillin molecule looks like a part of the bacterial cell wall that is cross-linked; that is,
4.,. PENICILLIN MOLECULE
Fig 4 - - U p p e r schematic representation shows how two parts o / c e l l wall /it on cross-linking enzyme (transpeptidase) just be/ore a cross-link is made. Lower picture shows how a penicillin molecule /its on cross-linking enzyme to block its action.
JOURNAL OF ENDODONTICS I VOI. 2, NO 5, MAY 1976
the penicillin molecule is a structural analogue for part of the bacterial cell wall 5-7 (Fig 6). It is believed that when a bacterial cell is dividing in the presence of penicillin, the crossqinking enzyme will "mistake" penicillin for ~he bacterial cell wall. The penicillin becomes irreversibly attached to the cross-linking enzyme, thus inactivating it. 5,7 As the bacterial cell divides and makes new bacterial wail, the crosslinking step cannot be completed and a weakened bacterial wall results.
Bacterial Spectra All bacteria thus far studied possess a murein component .in their cell walls. 3 Since the penicillins interfere with murein biosynthesis, why is it that all bacteria are not killed by penicillin; that is, why do the various penicillins have different bacterial spectra? There are two reasons. Some bacteria make an enzyme (penicillinase) that degrades the killer portion of the penicillin molecule 3 (Fig 7). This changes the shape of the penicillin molecule and it no longer is effective in blocking the transpeptidase enzyme. The penicillin is simply destroyed as fast as it can be introduced into the body. Therefore, an effective concentration of penicillin is never reached in the blood and tissues. The second reason is that the bacteria cell wall contains chemical components other than murein. These other components are responsible for the differential effect of the G r a m stain 2 and also prevent certain penicillins from penetrating to the transpeptidase enzyme. ~-5 Thus while penicillin G apparently cannot penetrate the outer portion (accessory portion) of the cell wall of gram-negative bacteria, ampicillin and carbenicillin can. Why? It has been suggested that by changing the modifier portion of the penicillin to a more polar moiety, such as in ampi-
151
JOURNAL OF ENDODONTICS [ VOL 2, NO 5, MAY 1976
cillin and carbenicillin, these molecules more readily penetrate the accessory portion of certain gramnegative bacteria, s
PENICILLIN G
METHI C I LLI N
Good "FIT"of Penicillin G on active site of Penicillinase
Poor "FIT" of Methicillin on active site of Penicillinase
Lack of T o x i c i t y of Penicillin Except in patients who are allergic to it, penicillin has no toxicity. This is because h u m a n cells lack a cell wall and a transpeptidase cross-linking enzyme, and thus penicillin has no action in mammalian cells. For this reason also, penicillin has no ,action on viral cells, which explains why penicillins are of no use in the ~reatment of viral infections such ,as colds and herpes simplex. Viral cells do not have a murein component and have no transpeptidase enzyme; thus there is no place for penicillin to act.
W h y Penicillin is B a c t e r i o c i d a l Bacterial growth in the presence of penicillin is really independent of penicillin. The bacteria will grow and divide and reproduce bacterial cell wall regardless of whether or not the cross-linking step is completed. If the bacterial cell can be maintained in an isotonic solution (that is, where the pressures both inside and outside the bacterial cell are equal), the bacterial cell will survive despite the weakened cell wall. In fac,t, this has been done experimentally; the cells are then called protoplasts. 1 Under normal conditions however, the pressures inside the bacterial cell are greater than outside; and when penicillin is effective, the cell wall and membrane ruptures, the contents of the bacterial cell spill out, and the bacteria cease to exist as living organized structures. P e n i c i U i n s t h a t Resist P e n i c i l l i n a s e Some penicillins such as methicillin are able to resist the action of peni-
152
cillinase, while others such as penicillin G are not. Since penicillinase opens the fl-lactam ring of the killer portion of the penicillin molecule, and both methicillin and penicillin G contain the fl-lactam ring, the question arises as to how methicillin escapes the action of penicillinase. Conceptually, we must speculate that methicillin has a modifier portion that prevents the killer portion of the molecule from fitting the active site of the enzyme properly. Because of this poor "fit" peniciUinase cannot catalyze the splitting of the fl-lactam ring (Fig 8). Summary The mechanism of action of the penicillins at the chemical level has been described. The reason why penicillin is not toxic to humans and viruses, the reason why different penicillins have different bacterial spectra, and bacterial resistance to penicillins also have been explained. Dr. Smith is a second-year endodontic resident at the Veterans Administration Hospital, Miami, Fla 33125. Formerly he was assistant professor of biochemistry and dentistry at .the dental school, University of Alabama in Birmingham. Reprint requests should be directed to Dr. Edwin L. Smith, Veterans Administration Hospital, 1201 NW 16th St, Miami, Fla 33125.
Fig 8---Good fit o/ peni. cillin on penicillinase is necessary for inactivation o] penicillin. Penicillinase-resis}. ant penicillins (such as methi. cillin) may escape action o] penicillinases because shape of modifier portion of penicillin molecule may prevent good fit.
References 1. Sharon, N. The bacterial cell wall. Sci Am 220:92 May 1969. 2. Lehninger, A.L. Sugars, storage polysaccharides, and cell walls, in Biochemistry. The molecular basis of cell structure and function. New York, Worth Publishers Inc., 1970, p 217. 3. Richmond, M.H., and Curtis, N.A.C. The interplay of /3-1actamases and intrinsic factors in the resistance of gram-negative bacteria to penicillins and cephalosporins. Ann NY Acad Sci 235:553 May 1974. 4. Boman, H.G.; Nordstr6m, K.; and Normark, S. Penicillin resistance in Escherichia coli K12: synergism between penicillinases and a barrier in the outer part of the envelope. Ann NY Acad Sci 235:569 May 1974. 5. Strominger, J.L. Enzymatic reactions in bacterial cell wall synthesis sensitive to penicillins, cephalosporins and other antibacterial agents, in Gottlieb, D., and Shaw, P.D. (eds), Antibiotics, Vol 1: Mechanism of action. New York, Springer-Verlag Inc., 1967, p 705. 6. Tipper, D.J., and Strominger, J.L. Mechanism of action of penicillins: a proposal based on their structural similarity to the acyl-D-alanyl-D-alanine. Proc Natl Acad Sci USA 54:1133 Oct 1965. 7. Strominger, J.L.; Willoughby, E.; Kamiryo, T.; BIumberg, P.M.; and Yocum, R.R. Penicillin-sensitive enzymes and penicillin-binding components in bacterial cells. Ann NY Acad Sci 235: 210 May 1974. 8. Butler, K.; English, A.R.; Ray, V.A.; and Timreck, A.E. Carbenicillin: chemistry and mode of action. J Infect Dis 122 (Suppl): S1 Sept 1970.
