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2 Penicillinase and Other β-Lactamases
Penicillinase and Other P-Lactamases NATHAN CITRI I . Introduction . . . . . . . . . A . Background . . . . . . . B. Definitions and Specificity . . . . C . Occurrence . . . . . . . . D . The Catalytic Reaction . . . . . I1. Molecular Properties . . . . . . . A . Purification and Physical Properties . . B. Composition and Sequence Analyses . . I11. Catalytic Properties . . . . . . . . A . Methods of Assay . . . . . . B . Kinetics and Substrate Specificity . . . C . Structural Modifications in Substrates . D . Structural Modifications in the Enzyme . E . Other Factors Affecting Activity . . . IV . Conformation and Function . . . . . . A . Nonspecific Conformational Transitions . B . Specific Transitions: Conformative Response V . Immunological Studies . . . . . . .
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23 23 25 26 27 27 27 31 35
35 39 40 41 42 44 44 45 46
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1 Introduction
A . BACKGROUND In 1940 Abraham and Chain ( 1 ) reported that an extract of Escherichia C O Z ~ destroys the antibacterial activity of benzylpenicillin . The authors presented evidence indicating that the extract (and possibly several 1. E . P. Abraham and E . Chain, Nature 146. 837 (1940).
23
24
NATHAN CITRI
other bacterial preparations) contained a penicillin-inactivating enzyme which they named penicillinase. Subsequently, many other bacteria were found to contain or secrete similar enzymes for which the inore general term, p-lactnmnse, Iins I ) c c i i suggesttxl. Tlicrc is 110 autliciiticntcd evidence that organisms other than bacteria produce p-lactamase ( 2 ) . The apparently exclusive and wide distribution of p-lactamascs anioiig diverse bacterial species suggests that this family of enzymes may have cvolvctl in connection with a uniquely bacterial function such as bacterial crll wall formation ( 2 6 ). The role of 1wnicillinasc and other ,8-luctnmascs i n conferring resistance to penicillins a i d the closcly relatctl cephalosporiiis has been amply demonstrated (6-8),niitl rcpcatcdly coiifirmctl in tlic numerous reports tlealing with the clinical aspects of p-lactmiase activity (reviewed in references 6, 7 , 9, 10).At a more fundaincntal lcvc~l,penicillinase has been studied most c.stcnsivcly from the point of view of enzyme induction ( 2 , 11, 1 2 ) , enzyme secretion (13-16), and transfer of genetic elements (17, 18). Earlier investigations a t the niolecular level, which have beeii reviewed in previous editions of this book (.19,20) , have been recently cbspanded to cover additional p-lactamase preparations. The picture which emerges indicates an unexpected diversity of size, structure, and molecular properties. Even more surprising is the variety of catalytic activities, which became increasingly evident as more p-lactamase preparations were iso2. N. Citri and M . R . Pollock. A ~ U U IEnzymol. I. 28, 237 (1966). 3. M. R. Pollock. Brit. M e d . J. IV, 71 (1967). 4. H. G. Boman. K. G. Eriksson-Grennberg, J . Foldes, and E. B. Lindstrom, in “Regulation of Nil[-lric A4cid and Protein Biosynthcsis” (V. V. Koningaherper and I,. 13osch. eds.). Vol. 10. 11. 366. B. B. A . Library, Elsevier Publ. Co., Amstcrdam. 1967. 5. I,. G. Burman, K. Nordstrom. and H. G . Bomnn, J. Bacteriol. 96, 438 (1968). 6. M. Barber, in “The Scientific Basis of Medicine,” Annual Review, 1964, p. 169. Univ. of London, (Athlone), London, 1964. 7. W. M. M. Kirby :ind R. J. Bulger. Ann. 12ee. M e d . 15, 393 (1964). 8. H. C. Neu, BBRC 32, 258 (1968). ‘3. E. Rauenbusch, Antibiot. Cliemolhercipia 14, 95 (1968). 10. J . T. Smith, J . M . T. Hamilton-Miller, nnd It. Knox, J . Phurm. Phurmtrcol. 21, 337 (1969). 11. M. R. Pollock, “The Enzyrncs,” 2nd ed., Vol. 1, p. 619. 1959. 12. M. H. Richmond. Essays Biocliem. 4 , 105 (1968). 13. J . 0. Lxnipen. J . Gen. Microbiol. 48, 249 and 261 (1967). 14. M. R. Pollock. J . Gcn. Microbiol. 26, 239 :ind 267 (1961). 15. D. J . Kushner and M. R . Pollock, J. Gett. Micl-obiol. 26, 255 (1961). 16. D. J . Kushner, J. Gen. Microbiol. 23, 381 (1960). 17. R. P. Novick, Bucteriol. Rev. 33, 210 (1969). 18. M. H. Richmond. A d v n n . Microbiol. Fhysiol. 2, 43 (1968). 19. E. P . Abraham. “The Enzymes,” Vol. 1, Pnrt 2, 1’. 1170, 1951. 20. M . R. Pollock, “The Enzymes,” Vol. 4, p. 269, 1959.
2.
PENICILLINASE AND OTHER
P-LACTAMASES
25
lated and studied against the larger number of scmisyiithetic penicillins and cephalosporins which became available in recent years.
B. DEFINITIONS A N D SPECIFICITY The term p-lactaubase denotes an enzymc which catalyzed thc hydrolysis of the amide bond in the p-lactam ring of 6-amino-penicillanic acid (6-APA) or 7-amino-cephalosporanic acid (7-ACA) and of their iV-acyl derivatives ( 2 ) .Such derivatives arc commonly referred to as penicillins [Fig. 1 (I) 1 and cephalosporins IFig. I (III), (V), and ( V I I ) ] , respectively. There is no evidence that any bond other than thc ainide bond in the intact nucleus of penicillin or cephalosporin is broken by the
(V11)
(VIII)
FIG.1. General structure of substrates tind products of the p-lactamase reaction, Substrates: (I) penicillins; (111). ( V ) , and ( V I I ) rephalosporins. Products: (11) penicilloic acids; ( I V ) , (VI), and (VIII) cephalosporoic acids.
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NATHAN CITRI
enzyme ( 2 1 ) . Thus the catalytic specificity of p-lactamases appehrs at present to be confined to these two closely related families of antibiotics. The term penicillinuse has been retained in common usage to denote p-lactamase preparations which predominantly catalyze the hydrolysis of penicillins. Conversely, preparations which are more active against cephalosporins have been frequently referred to as cephulosporinases. It is expected that this distinction will disappear as more p-lactamases are found to show a continuous spectrum of activity against penicillins and cephalosporins.
C. OCCURRENCE Although @-lactamaseis widespread among the various bacterial species ( 2 , 9 ) ,the levels of p-lactamase activity within each species are extremely variable. The range (22) and significance of such variations have been the subject of a recent discussion by Pollock ( 2 3 ) . Indeed, studies prompted by variations in the penicillinase-forming ability of bacteria led to important advances in our understanding of the transfer and control of genetic information. In the present context the following observations may be relevant: (1) The gene for p-lactamase is readily lost (or acquired) in strains of bacteria (e.g., staphylococci and enteric bacteria) (24) where it is carried by an extrachromosomal particle (18,2 6 ) . (2) The gene expression (i.e., rate of p-lactamase formation) is often controlled by the mechanism of induction ( 2 , 1 1 , 2 7 ) . (3) A single mutational event, resulting presumably in the replacement of a single amino acid residue, may cause a large decrease in activity (3, 23). 21. The suggestion has been made that certain peptides which have been studied as inducers of p-lactamase in Bacillus cereus and in S. atireus may serve as substrates for the enzyme [A. K. Saz and D. L. Lowery, BBRC 15, 525 (1964); 19, 102 (1965)l. However, there is no evidence that such peptides are in fact hydrolyzed by plactamase. 22. J. M . T . Hamilton-Miller, FEBS Letters 1, 86 (196S). 23. M. R . Pollock, Ann. N . 1’. Acncl. Sci. 151, 502 (1968). 24. In enteric bacteria the p-lactamase gene may be acquired by infection with an R factor (cf. Table IV). This led to the pertinent observation (25) that no plactamase should be regarded as “species-specific” until its gene is shown to bc rhromosomal. 25. N. Datta and M. H. Richmond, BJ 98, 204 (1966). 26. R . P. Novick, Ann. N . Y . Acad. Sci. 128, 165 (1965). 27. M . H . Richmond, Nature 216, 1191 (1967).
2.
PENICILLINASE AND OTHER
P-LACTAMASES
27
D. THECATALYTIC REACIYON By definition, p-lactamase catalyzes the hydrolysis of the P-lactam ring in penicillins or cephalosporins as shown in Fig. 1. The product of the reaction with a penicillin has been identified by Abraham et al. (28) as the corresponding penicilloic acid. Although few other cases have been as rigorously investigated, it is generally accepted that the action of a p-lactamase on a penicillin results in the formation of a relatively stable, single product, penicilloic acid [Fig. 1 (11) 1. An analogous reaction has been postulated for cephalosporins (29,30), but it soon became apparent that the presumed primary product is highly unstable (31, 32) except in the case of cephalosporin y-lactones [Fig. 1 (VII) and (VIII)]. In other cephalosporins the nature of the primary product appears to depend on the substituent R [Fig. 1 (III)] (33).When R’ = H, an unstable “cephttloeporoic acid” is formed [Fig. 1 (V) and ( V I ) ] which is a product analogous to the penicilloic acid. In contrast, when R’ is an acetoxy or pyridinium group, the reaction involves the loss of R’ with the formation of an exocyclic double bond and a rearrangement as shown in Fig. 1 (IV). The resulting compound (Amax = 230 nm) is unstable and undergoes fragmentation (33). II. Molecular Properties
A. PURIFICATION AND PHYSICAL PROPERTIES The numerous reports of p-lactamase activity in various bacterial strains reflect the widespread occurrence and the clinical importance 28. E. P. Abraham, W. Baker, W. R. Boon, C. T. Calam. H. C. Carrington, E. Chain, H. W. Florey, G. G. Freeman, R. Robinson, and H. G. Saunders, in “The Chemistry of Penicillin” (H. T. Clarke, J. R. Johnson, and R. Robinson, eds.). Chaptrr 2. p . 10. Princeton Univ. Press, Princeton, New Jersey, 1949. 29. B. Crompton. M. Jago, K. Crawford, G . G. F. Newton, and E. P. Abraham. BJ 83, 52 (1962). 30. G. G. F. Newton and E. P. Abraham, Nature 175, 548 (1955). 31. L. D. Sabath, M . Jago, and E. P. Abraham, BJ 96, 739 (1965). 32. G. G. F. Newton, E. P. Abraham. and S. Kuwabara, Antimicrobinl Agents
Chemothempy p. 449 (1968). 33. J. M. T. Hamilton-Miller, G . G . F. Newton, and E. P. Abrahani, BJ 116, 371 (1970). 34. Similarly, two distinct p-lactamasee have been observed in pure cultures of Enlerobncter ( 3 6 ) . 35. T. D. Hennessey, J . Gen. Mksobiol. 49, 277 (1967).
TABLE I PURIFIED ~LACTAMASE PREPARATIONS Source of enzyme 1. Bacillus cereus 569/H
extracellular (8-lactamase I) 2. Bacillus cereus 569/H extracellular (8-lactamase 11) 3. Bacillus crrrus 569/H cell bound (8-lactamase 7 ) 4. Bacillus cereus 5/B
ex tracellular
5. Bacillus licheniformis 749/C eutracellular 6. Bacillus licheniformis 749/C cell bound
7. Bacillus lirheniformis 6346/C extracellular and cell bound
Main purification steps 1. Selective adsorption on glass 2. Ammonium sulfate fractionation 1. Ammonium sulfate fractionation 2. Ethanol precipitation 1. Sonic disintegration 2. Selective adsorption on cellulose phosphate 3. 1. CJI-cellulose chromatography Acetone precipitation 2. Ammonium srilfate fractionation 3. 1. Ethanol precipitation Selective adsorption on cellulose phosphate 2. Ammonium sulfate fractionation 1. Lyxozyme and trypsin digestion 2. Ammonium srilfate fractionation 3. DEAIGcelliilose chromatography 1. Ammonium sillfate fractionation
Criteria of homogeneity
Ref.
Electrophoretic and sedimentation patterns Crystallized
36
ChZ-cellulose rechromatography
38
Electrophoretic and sedimentation patterns
39
Sedimentation pattern
40
DEAEcellulose rechromatography
40
Electrophoretic and sediment,ation pat terns
40
37
8. Staphylococcus aurcus (types A, B, and C) extracellular 9. 10.
11. 12. 13. 14.
a
1. Selective adsorption on
cellulose phosphate 2. CM-cellulose chromatography 3. Centrifugation on sucrose gradient 4. Electrophoresis on sucrose gradient 1. Ultrasonic disintegration Escherichia coli TEM (R-TEM)o 2. DEAEcellulose chromatography cell bound 3. Gel filtration Escherichia coli K-12 (Glla1)b 1. Release by spheroplasting 2. SE-cellulose chromatography cell bound 3. Chromatography on hydroxylapatite 1. Osmotic release Escherichia coli DB103 (ATSm)a 2. DEAE-cellulose chromatography cell bound 3. Selective adsorption on hydroxylapatite Escherichia coli W3630 ( R G N Z ~ ~ ) ~1. Ultrasonic disintegration 2. DEAE-cellulose chromatography cell bound 3. CM-cellulose chromatography 1. Ultrasonic disintegration Eschwichia coli W3630 (RGN14). 2. Streptomycin precipitation cell bound 3. DEAE-cellulose chromatography Enterobacter cloacae 214 1. Ultrasonic disintegration cell bound 2. Chromatography on CM-Sephadex G-50 3. Gel filtration
CM-cellulose rechromatography ; sedimentation patterns
DEAE-cellulose rechroma tography
41
25,42
Electrophoretic and sedimentation patterns; immunodiff usion Single band on gel electrophoresis
43
Electrophoretic and sedimentation patterns
44
Electrophoretic and sedimentation patterns
44
Electrophoretic pattern and CM-cellulose rechromatography
45
The 8-lactamase gene was acquired by infection with the R factor shown in parentheses. The 8-lactamase gene is chromosomal.
8
30
NATHAN CITRI
of this group of enzymes. However, in most cases no attempts were made t o isolate the enzyme and the available information is too fragmentary to permit characterization a t the molecular level. Table I lists p-lactamase preparations which have been purified and shown to be homogenous. The different purification procedures, summarized in Table I (25,34-45),reflect to some extent differences in the cellular location and in the physical properties of the various preparations. Of particular interest is the isolation of distinct p-lactamases from a single source such as Bacillus cereus strain 569/H, which yields the three P-lactamase preparations listed in Table I (34).This strain is a constitutive mutant of the inducible strain 569 of B. cereus which produces similar levels of all three variants of p-lactamase upon induction (46-49). The relationship between the cell-bound (7-penicillinase) variant and p-lactamase I (a-penicillinase) has been extensively investigated (38, 46, 50-54) but not clarified. Much less is known about the more recently discovered p-lactamase I1 in relation to the two other varieties. One fascinating question which remains open is the interconvertibility of the three varieties (2,.25,37,38). According to a recent report ( 5 5 ) , p-lactamase I of strain 569 can be further resolved by P-cellulose chromatography. The three fractions thus obtained appear to retain their identity on gel electrophoresis, even in the presence of urea, and yield distinctive tryptic digest maps.
36. M. Kogut, M. R . Pollock, and E. J. Tridgell, BJ 62, 391 (1956). 37. S. Kuwabara, and E. P. Abraham, BJ 103, 27C (1967). 38. N. Citri and A. Kalkstein, ABB 121, 720 (1967). 39. M. R. Pollock, A-M. Torriani, and E. J. Tridgell, BJ 62, 387 (1956). 40. M. R. Pollock, BJ 94, 666 (1965). 41. M. H. Richmond, BJ 94, 584 (1965). 42. N. Datta and P. Kontomichalou, Nature 208, 239 (1965). 43. E. B. Lindstrom, H. G. Boman, and B. B. Steele, J. Bucteriol. 101, 218 (1970). 44. S. Yamagishi, K. O’Hara, T. Sawai, and S. Mitsuhashi, J. Biochem. (Tokvo) 66, 11 (1969). 45. T. D. Hennessey and M. H. Richmond, BJ 109, 469 (1968). 46. M. R. Pollock, J . Gen. Microbid 15, 154 (1956). 47. L. D. Sabath and E. P. Abraham, BJ 98, 11C (1966). 48. R. Sheinin, J. Gen. Microbial. 21, 124 (1959). 49. J. D. Duerksen, BBA 87, 123 (1964). 50. J. D. Duerksen and M. L. O’Connor, BBRC 10, 34 (1963). 51. M. R. Pollock and M. Kramer, BJ 70, 665 (1958). 52. N. Citri, BBA 27, 277 (1958). 53. N. Citri, Bull. Res. Council Israel A9, 28 (1960). 54. E. Ron-Zensiper and N. Citri, Nature 198, 887 (1963). 55. J. Imsande, F. D. Gillin, R . J. Tanis, and A. G. Atherly, JBC 245, 2205 (1970).
2.
PENICILLINASE AND OTHER
p-LACTAMASES
31
Several molecular properties of purified P-lactamase preparations are listed in Table I1 (2,25,36,39,40,41,@, 56-58).I n most cases reported the molecular weight is within the range of 28,000-30,000, which seems to be typical for the gram-positive p-lactamases. Similar values have been reported for some but not all, gram-negative p-lactamases. Thus, the values for the enzymes from several strains of E . coli and Salmonella typhinzurium are in the range of 22,00032,000 (43,44, 59,60).In contrast, lower values (14,00C)-17,000) have been reported for other J3-lactamase preparations isolated from E . coli (Table 11, 26, 4 )and from a strain of Enterobacter cloacae (45). The interesting suggestion has been made (61) that the unusually high molecular activity of extracellular p-lactamases (Table 11) may possibly compensate for the dilution of the secreted enzyme in the growth medium. Partial data on the optical rotation (57)and optical rotatory dispersion (55)of native, denatured, and renatured /3-lactamases of B. cereus suggest fairly strong hydrophobic interactions and compact folding. An a-helix content of 30% has been proposed for the native and renatured enzyme of B. cereus 569 (66).
B. COMPOSITION AND SEQUENCE ANALYSES The amino acid composition of the p-lactamases listed in Table I11
(2, 40,41, 43, 45,54, 55, 58, 62-64) underlines the similarities between
preparations derived from closely related strains of bacteria, especially if the combined basic and acidic amino acid content is compared ( 2 3 ) . The staphylococcal enzymes are remarkably rich in lysine, and the lysine-arginine content of the other gram-positive p-lactamases is almost as high. Complete absence of cysteine appears to be characteristic (2,55), although J3-lactamases which contain a t least one cysteine residue have been reported (37,65,66). 56. J. R. Hall and A. G. Ogston, BJ 62, 401 (1956). 57. N. Citri, N. Garber, and M. Sela, JBC 235, 3454 (1960). 58. M. H. Richmond, BJ 88, 452 (1963). 59. C. Lindqvist and K. Nordstrom, J. Bacterial. 101, 232 (1970). 60. H. C. Neu and E. B. Winshell, ABB, 139, 278 (1970). 61. M. R. Pollock, in “The Bacteria” (I. C. Gunsalus and R. Y . Stanier, eds.), Vol. 4,p. 121. Academic Press, New York, 1962. 62. S.Jacobs, Res. Commun. 16th Congr. Quim. Anal., Lisbon, 1966 p . 212 (1956). 62a.M.H.Richmond, personal communication (1966). 63. R. P.Ambler and R. J. Meadway, Nature 222, 24 (1969). 64. R. J. Meadway, BJ 115, 12P (1969). 65. J. T.Smith, Nature 197, 900 (1963). 66. L. D.Sabath and M. Finland, J. Bacterial. 95, 1513 (1968).
TABLE I1 MOLECULAR PROPERTIES OF PURIFIED &LACTAMASES
Source of enzyme"
B. cereus 569 B. cereus 569/H (1) B. cereus 5/B (4) B. lichenifomis 6346 (7) B. Zicheniformis 749/C (6) B. Zicheniformis 749/C (5) S.nureus A ( 8 ) S. aureus B (8) S . aureus C (8) E. wli TEM (9) E. coli G l h l (10)
Molecular weight 31,500 30,800 35,200 28,000 28,000 28,100 29,600 29,600 29,600 16,700
29,OOO
Molecular activityb 1.60 X 1.53 X 1.48 X 2.10 X 1.18 x 1.08 x 2.0 x 2.8 X 1.9 X 2.0 X 2.08 X
106 1W 106 lW 106 106 104 lo3 l(r 10'
1Oa
Sedimentation coefficient (10'8 X ~ 2 0 2.68e 2.5-2.65" 2.82~ 2.63 2.70 2.66 2.62 2.5 2.5 1.85 3.4
EE
, ~ )
(nm/mg N) 6.0 6.35 7.35 5.65 5.45 4.75 7.38
Electrophoretic mobility0
(cm*/sec/VX
Asc., 1.68; Desc., 1.70d Asc., 1.73; Desc., 1.65d
Desc., 3.4@ Asc., 4.95; Desc., 4.50/ Asc., 4.33; Desc., 3.65/ -
-
13.1
-
~
Numbers in parentheses are the serial numbers of the preparations in Table I. Moles benzylpenicillin hydrolyzed per mole enzyme per minute at 30". The diffusion coefficients determined (66) for preparations 569, 569/H and 5/B are 8.28, 8.38, and 7.80 (107 x D m J . In glycine buffer, p = 0.2, pH 8.4. e In glycine buffer, p = 0.2, pH 8.15. In veronal buffer, p = 0.1, pH 8.15. u Aw., ascending; l)esc., descending. a
b
Ref.
10-6)
36, 66 36, 66, 67 36, 39, 66 2, 40 2, 40 40 68
41 41 26
43
TABLE I11 AMINOACID COMPOSITION OF B-LACTAMASES E. coli E. cloacae Amino acids Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Cysteine WPhPhan N-term AA Total ammonia amide
214 G l l a l D3lo (Ref. 43) (Ref. 46) 9 2 5
10 8
7
12 9 10 15 10 5 6 10 5 3 ? ?
17 4 10 25 18 16
33 19 23 28 17 4 15 23 10 8 0
-
17 4 9 23 18 14 32 20 22 29 17 5 17 24 12 7 0 6 Ala (22)
B. cereus 5/B
B. lichenijormis
569 569/H (Ref. 2, 62)
31 23 6 6 13 11 33 25 9 12 2 4 25 22 6 10 19 21 30 29 17 15 5 1 20 22 17 19 5 7 7 8 0 0 Aspb Lysc
6346/C (Ref. 40)
749/c (Ref. 63, 64)
21 5 10 31 13 5 22 10 19 33 17 1 22 18 4 6 0
19 1 12 28 18 10 24 9 12 19 10 5 9 21 5
24 1 15 37 21 11 27 11 15 26 15
5
-
LYS
7 3 Lysd (20)
-
0 -
5 14 27 6
S. a u r m TypeA TypeB (Ref. 64,68) (Ref. 4 1 ) 43 2 4 39 13 19 18 9 12 18 16 3 19 22 13 7 0 2 LYS (29)
43 2
TypeC (Ref. 4 1 )
42 12 17 22 11 16 21 11 2 15 22 9 7 0 -
45 3 4 39 11 17 18 10 15 19 11 2 18 23 14 7 0 -
(28)
LYS (32)
5
-
The &Iactamase gene was received from G l l a l and both eneymes were purified by identical procedures (Serial No. 11 in Table I). Richmond (@a). c Imsande et al. (66). d See, however, text and Ambler and Meadway (63). a
w
TABLE IV OF VARIOUSSUBSTRATES (RATEOF HYDROLYSIS OF BENZYLPENICILLIN = 100) RELATIVE RATESOF HYDROLYSIS
I@
~
~~
Substrates
Source of enzyme"
Phenoxymethyl- Ampipencillin d i n
Bacillus licheniformis Strain 749/C (3 isozymes) [5] (mutant 77) Strain 6346/C [7] Bacillus cereus Strain 569/H 8-lactamase I [I] plactamase I1 [2] 8-lactamase y [3] Strain 5/B [4] Staphylowccus aureua Types A and B [8] Type C I81 Escherichia w l i Strain TEM (R-TEM) [9] Strain K-12 (Gllal) [lo] Strain W3630 ( h N Z 3 8 ) [I21 Strain W3630 ( h14) ~ [I31 Enlerobacler cloacae 214 [14] a
-
153
64-74
-
-
Oxacillin
Cloxacillin
0.45-0.50 4.0 1.0
-
-
-
4.6-5.0 5.0 13
0.7 89 <0.1 -
40 9
3.5 1.0
10 -
0.5 1.2
-
5 89 5 4
6-APA
120 64 55
-
3.0 89 26 1.8
100 100
-
1.5 0.6
4.5 1.0
50 -
1 -
-
-
110 4 450
-
-
27 292
-
140
-
-
-
130
86
11
-
-
-
-
-
171 96
Cephalosporin C
Methicillin
-
Numbers in brackets are the serial number of the preparation in Table I.
85 1.2 263
Benzylcephalosporin
0.3-0.4 0.9.5-1.06 11.0 15 42
17.5-19.8 275 -
Ref.
23 23 40
3 41 17 -
38, 75 37 38 29, 76
-
-
41,58
0.8 920 -
16 -
65 72 3.6
44
-
-
69
44
1830
8200
45
80
0.02
7200
<0.1 -
Cephaloridine
-
41 25 43
f
ael
E
2.
PENICILLINASE AND OTHER
p-LACTAMASES
35
The data on terminal amino acids (Table 111) support the accepted notion that every p-lactamase consists of a single polypeptide chain. There are no indications to the contrary, although firm evidence is lacking in most cases. Further observations pertaining to the chemical composition of plactamases and comments on the significance of the reported variations can be found in recent reviews ( d , S , 23). Ambler and Meadway have very recently reported on their studies of the amino acid sequence of p-lactamases from representative strains of S. aureus and B. lichenijormis (63, 6 4 ) . The complete sequence of the p-lactamase protein of S. aureus PCI (type A) was determined by the characterization of the peptides produced by digestion with trypsin, chymotrypsin, or pepsin. The sequence analysis of the p-lactamase protein of B . licheniformis 749/C was performed on both the extracellular and the cell-bound enzyme (cf. serial Nos. 5 and 6, Table I ) . The slight differences between the two forms were confined to the N-terminus and attributed to the difference in the release mechanism. Peptides corresponding to about 90% of the molecule have been characterized and their sequences combined to form five larger fragments. The results of these studies are summarized in Fig. 2, which presents the S. aureus sequence and the B. lichenijormis fragments arranged in such a way as to give the best possible match with that sequence. This arrangement implies that the B . licheniformis molecule is several residues longer a t each end (see also Table 111).The authors estimated that the complete sequence of the B. lichenijormis protein will show about 40% identity with that of S. aureus. This estimate has been most recently confirmed (64), and the homology proposed in Fig. 2 is now firmly established. There can be little doubt that both proteins share a common origin and that their divergent evolution accounts for the differences in the catalytic properties listed in Tables IV and V.
111. Catalytic Properties
A. METHODSOF ASSAY The methods most commonly used for the quantitative assay of plactamase are based on one of the following principles: (1) Manometric or alkalimetric titration of the new carboxyl group formed by hydrolysis of the p-lactam ring; (2) iodometric titration of the product, and (3)
1
8
Lys-
Thr-
26 27 -THR- Lss-Thr-
3
4
5
Glu- Met- Lys28
6 7 Lys- Glu-
Asp- Asp-
8 9 10 11 12 13 14 15 Leu- Asn- Asp- LEV- GLU- Lys- Lys- TyrPhe-
Lys-
LD Leu-
Glu-
Glu-
Glu- Phe-
Ser-
29 Gly-
30 Lys-
Glu-
32 VAL-
Gly-
Thr-
Asn- Arg-
Thr-
Val-
Ah-
Tyr-
Arg-
LC Pro- Asp-
Glu-
51 -Ser-
5’2 Ala-
53 Ile-
54 55 56 Leu- Leu- Glu-
5i Gln-
58 Val-
59 Pro-
60 Tyr-
61 62 Asn- Lys-
63 64 65 Leu- Asn- Lys-
Ser-
Ile
(Glx,
GIx,
Asx.
76 -Ala-
79 80 81 87 83 84 85 86 TYI:- Ser- PRO- ILE- Leu- GLT- LYS- Tyr- VAL- GlyLE Tyr- Asn- Pro- IleThr- Glu- Lys- His- Val- Asp-
87
38 89 Asp- Ile-
31
33 34 35 LD- Phe- Am-
Lys-
8
Ah-
-Val-
??
36
Ser-
id
16 17 18 Asn- ALA- His-
19 He-
20 21 GLT- Val-
Ah-
Leu-
Gly-
Asp-
57 38 39 40 41 42 ASP- Lys- ARG- PHE- ALA- Tyr-
Lys-
Arg-
Asx,
Phe- Ala66 Lys-
L e u ) Arg-
-Tyr-
Jer
126
127 -Oh- Leu-
Ile-
(Asp.
Asti) Ala-
128
199 130 G L P - ASP- Lys-
106
Gln-
Asn-
131 13‘7 13) VAL- Thr- Asn-
LCJ- Ile-
112 113 LPS- GluLB Leu- Lys- Gln-
134 Pro-
135 Val-
136 137 AKG- Tyr-
Gly-
Asp-
0111- Val
Asx,
Pro,
Glx)
Arg-
Phe-
151 132 153 .Asp- TIIR- Ser-
154 Thr-
155 Pro-
15G Ala-
157 158 ALA- I’he-
159 Gly-
160 Lys-
161 TI r-
162
Srr)
Ala-
Are
Ala-
Val-
Thr-
Scr-
-Lys-
Asx)
Thr
(Tlir,
(Thr.
Leu-
44 45 46 43 ALA-, S E R - T H R - Ser-
Phe
Ah-
Leu- Asp
48 49 47 LTS- ALA- IleAla-
50 Am-
Leu
Ser-
Thr-
Ilc-
Lp-
68 His-
69 Ile-
70 Asn-
71 Lys-
73 74 ASP- ASP- Ile-
75 Val-
Thr-
Tyr-
Thr-
Arg-
Asp-
Asp-
Leu-
9.5 96 Ile- Glu-
07 Ala-
08
Ser-
99 100 Met- Thr-
67 ValLE Ile-
91 92 93 THR-LEU- LTS- Ala90
94 LEr-
7‘7
Asn-
Thr- Gly- Met- Thr- Leu- Lys
107 10rl 109 110 111 ALA- Asn- A S S - L ~ s - I L E - IleAh-
Ile-
23 24 !25 AbA- LEU- ASP-
Phe- Ala-
Ser!
Leu- Arg.
103 Arg-
124 125 LEV- Lya-
Pro)
Lru-
Glu-
Leu- Arg-
144
145 Ser-
146 Pro-
147 L?s-
148 Stx-
149 Lys-
150 Lys-
(GI?,
Pro,
Thr.
1;1x, Asx,
Asr.
Leu- Ala
118 Lys-
119 Val-
Glu
(Ser,
140 141 142 143 GLU- LEU- Asn- Tyr-
Ile-
138 139 GLU- IleLB Glu- Pro163
Ah,
120 121 122 LJ-s- LTS- Gin-
Glu-
114 115 116 117 ILE- GLP- GLT- Ile-
Lys-
102 103 104 105 -TYR- S E R - Asp- Asn- Thr101
Lys-
Pi! Tyr-
Gly-
Glu-
GI?-
Leu-
1G4 165 166 LEU- A m - Lys- Leu- IleLB Leu- A r e 41a- Phe- A h -
Tyr-
Gln-
Glu-
Val
16T Ah-
168 Asn-
160
Leu-
Glu-
(Asp.
Lys- Lys-
17‘3
174
GI!--
1TO 171 179 LTS- LEU- Ser-
L?-S.
4:
Asp-
LXS- Leo-
Ser-
Glu-
Pro-
175
1.1.- ASU
Lys-
176 - L p
IT7
178
Lys- Plie-
179 180 LEV- Leu-
181 182 ASP- Leu-
NET- Leu- Asn- Asn- Lys- Ser-
183
Leo-
Asp- Trp-
Met-
184
185
186
187
188
1R9 Gly-
190 Asp-
191 Tlir-
192 193 194 LEV- ILE- Lyr-
195 Asp-
196 197 GLT- Val-
Pro-
199 Lys-
200 Asp-
Asn)
Leu- Ile-
Arg-
-4Ia-
Gly
(Asp,
Glu,
Pro.
Trp.
217 .4sn-
919 Val-
220 Ma-
5'11 Phc-
222 Val-
223 224 Tyr- Pro-
225 Lyn-
249
250
193
LB
Lys- Arg- Asp (Thr. Thr,
Leu-
201 202 -Tyr- Lys-
203 204 Val- Ala-
205 206 207 208 209 ASP- LTS- Ser- CLY- Gln-
Gly,
Ala.
Val,
Val)
Asp-
226
227
228
Gln-
Ser-
229 (;hi-
230 231 Pro- Ile-
-lily.
252 ILE- Ser2.51
-1lew
Ile-
-Are Glu-
Ala-
253 254 GLU- Thr-
Glu-
Ah-
255 Ah-
Thr-
Lys- Thr232 Val-
210 211 ALA- IleLB
Ala-
Gly,
212 213 Thr- Tyr-
214 Ala-
215 216 Ser- Arg-
Thr-
(ily-
Ala-
Ala-
Ber-
Tyr-
Cly-
Arg- Asn- Asp- Ilc-
Aln
233 Leu-
234 Val-
235 Ile-
236 Phe-
237 Thr-
238
239 Asn- Lys-
240 241 ASP- Asn-
242 Lys-
244 Asp-
Ala-
Val-
Leu-
Ser-
Ser-
Arg-
Asp
(Ma,
Lys,
256 257 LYR- Srr-
258 259 260 961 VAL- MET- LYS- Glo-
252
263
264
265
266
967
Phc
Lys-
LA Val-
Leu- Asn-
Met- Asn-
Gly-
Val-
218 Asp-
Met-
Lys- Ala-
243 SerLA Asp,
245 246 LTS- Pro-
L?-s) 1 ~ s - Tyr-
247
248
b s n - A>I'- LYS- LEUAsp-
ASP- Lys-
Leu-
Lys
FIG.2. Amino acid sequences of P-lactamases of Staphylococcus aureus PCI (upper line) and of Bacillw licheniformis 749/C (fragments LA to LE, lower line). Matching residues are shown in block letters (upper line). (From Ambler and Meadway, 63.)
TABLE V APPARENTDISSOCIATION CONSTANT^
(pM)
Substrates Source of enzyme" Bacillus licheniformis Strain 749/C (5) Strain 6346/C (7) Bacillus cereus Strain 569 p-lactamase I* Strain 569/H 8-lactamase I (1) p-lactamase I1 (2) Strain 5/B (4) Staphylococcus aureus Type A (8) Type 80/81b Escherichia coli Strain 214 Tb Strain TEM (9) Strain W3630 ( F ~ 238) N (12) Strain W3630 (&N 14) (13) Aerobacter cloacaeb Pseudomanas pyocyanea
Benzylpenicillin 49 9.5
Ampicillin -
-
Methicillin 0.93 0.23
Oxacillin
Cloxacillin
-
-
b
16.6 16.7
Cephalosporin C 50 50
Cephaloridine
Ref.
-
48
110
410
550
-
-
-
-
60 3300 43
1300 3300
-
460 1600 230
570 1600 330
2300 -
3000 -
-
1100 -
3300
-
77, 79, 80 37 20, 77
5 12
170
16000 5000
5000 -
-
120
100 -
-
41, 58, 81
22 5.1 27.8 8 13
35oc 2.3 15.9 34.0 541 -
>46& 22 26.3 222
-
-
-
1000
600 111 400 69 130
44= 3.2
-
-
-
0.lP
1.lC
-
0.g 13.0 16.5c 0.026
Numbers in parentheses are the serial numbers of the preparations in Table I. For purification procedure see original references. c Based on KI with benzylpenicillin as substrate. a
6-APA
-
-
46, 77, 78
78 82, 83
25
44 44
83 31
2.
PENICILLINASE AND OTHER
P-LACTAMASES
39
colorimetric determination of the hydroxaniate formed by residual substrate. The various applications of these and several other principles have been reviewed elsewhere (67, 6‘8).The assay methods, primarily designed for benzylpenicillin, the standard substrate, are generally suitable for other substrates, although in some cases the procedure has to be modified. Specific problems involved in the replacement of benzylpenicillin with other substrates, especially cephalosporins (69) have been recently discussed ( 2 , 31, 32, 7 0 ) . )
B. KINETICSAND SUBSTRATE SPECIFICITY
It is generally agreed that the &I-lactamase reaction is of zero order in the presence of saturating substrate concentration (71) and that the enzyme displays typical Michaelis-Menten kinetics over a wide rangc of concentrations (72). The slight deviations, which have been observed with crude enzyme preparations only (59, 73) deserve further investigation ( 4 ) . Anomalously high dissociation constants recorded with certain substrate combinations (74) have been correlated with the effect of such substrates on the conformation of p-lactamase (see Section IV,B) . Individual “substrate profiles” of variously related p-lactamases and apparent dissociation constants for several substrates are listed in Tables IV (2.9)25, 29,37, 38,40, 41, 43-45, 58, 75, 76) and V (20, 25, 31, 37, 40) 41, &,46,58,76,77--83), respectively. An interesting attempt to correlate )
67. J. M. T. Hamilton-Miller, J. T. Smith, and R. Knox, J. Pharm. Pharmacol. 15,81 (1963). 68. N. Citri, Methods M e d . Res. 10, 221 (1964). 69. E. P. Abraham, Phamtacol. R e v . 14, 473 (1962). 70. M. R. Pollock, J. Fleming, and S. Petrie, Proc. 2nd Meeting Federation Euro-
pean Biochem. SOC.,1966 p. 139. Pergamon Press, Oxford, 1967. 71. R. J. Henry and R. D. Housewright, JBC 167, 559 (1947). 72. J. E. Banfield, Experientia 13, 403 (1957). 73. W.Rothe, Pharmazie 5, 25 (1950). 74. N. Zyk and N. Citri, BBA 151, 306 (1968). 75. N. Citri and N. Zyk, BBA 99, 427 (1965). 76. S. M. Chaikovskaya and T. G. Venkina, Antibiotiki 9, 329 (1964). 77. N. Citri, N. Garber, and A. Kalkstein, BBA 92, 572 (1964). 78. R. H. Depue, A. G. Moat, and A. Bondi, ABB 107, 347 (1964). 79. F. R. Batchelor, J. Cameron-Wood, E. B. Chain, and G. N. Rolinson, Proc. R o y . SOC.B154, 514 (1961). 80. S. Kuwabara and E. P. Abraham, BJ 115, 859 (1969). 81. M. H. Richmond, Brit. M e d . Bull. 21, 260 (1965). 82. J. M. T. Hamilton-Miller and J. T. Smith, Nature 201, 999 (1964). 83. J. M. T. Hamilton-Miller, J. T. Smith, and R.Knox, Nature 208, 235 (1965).
40
NATHAN CITRI
these values in a physiologically meaningful way has been made by Pollock (40), who pointed out that under typical ecological conditions the enzyme functions at unsaturating substrate concentrations. Thus the “physiological efficiency” of a p-lactamase depends on both the V,,,,, and the K , value and may be conveniently expressed as the ratio VlnaJKm. There can be little doubt that this ratio is an important parameter in comparing 8-lactamases in any context dealing with the ecology, evolution, and physiological significance of the enzyme (2,25,40,84).
C. STRUCTURAL MODIFICATIONS IN SUBSTRATES The effect of replacement of the APA nucleus with an ACA nucleus on the susceptibility of the substrate to enzymic hydrolysis depends on the nature of the p-lactamase ( 2 ) . It has been richly illustrated in the case of the p-lactamase of Pseudomonas pyocganea (31)and analyzed in terms of conformative response (see Section IV,B) in a study of a B . cereus p-lactamase (75).I n that study the role of the nucleus was examined in both catalytic and noncatalytic enzyme-substrate interactions. Little has been published on the effect of modifications within each nucleus, presumably because such modifications lead, as a rule, to the loss of antibiotic activity. Conversion of the carboxyl groups in penicillins confers partial resistance to p-lactamase (85-87).I n cephalosporins, replacement of the acetyl group in position 3 of the dihydrothiazine ring [ R in Fig. 1, (111)] by pyridine causes increased susceptibility to hydrolysis by p-lactamases of Pseudomonas pyocyanea (31,88),Enterobacter cloacae (45),E . coli (42,88),and both the extracellular and cell-bound enzymes of B . cereus (38)* The most common modification is the replacement of the N-acyl substituent (“side chain” R in Fig. 1 ) , especially in penicillins. Indeed, the differences in K , and relative V,,, values among the penicillins listed in Tables IV and V all result from the differences in the nature of the side chain. Analysis of such data (Table VI) (25,@, 4,70,76,77-79, 81, 89,901 84. M. R. Pollock, Antimicrobial Agents Chemotherapy p. 292 (1965). 85. D. E. Cooper and S.B. Brinkely, JACS 70, 3966 (1948). 86. N . J. Huang, T. A. Seto, J. M. Weaver, A. R. English, T. J. McBride, and G. M. Schull, Antimicrobial Agents Chemotherapy p. 493 (1964). 87. E. Grunberg and G . Beskid, Antimicrobial Agents Chemotherapy p. 619 (1968). 88. L. D. Ssbath and M. Finland, Ann. N. Y. Acad. Sci. 145, 237 (1967). 89. R. P. Novick, BJ 83, 229 (1962). 90. J. M. T. Hamilton-Miller, BJ 87, 209 (1963).
2.
41
PENICILLINASE AND OTHEB ~-LACTAMASES
TABLE VI EFFECTOF SIDE CHAINON CATALYTIC CONSTANTS OF PENICILLINS Side chain Phenylacetyl-
V,..:: V,..P 5.3 16.7 2.6 20.0 10.0
Phenylglycyl-
Dimethoxybenzoyl-
K,r: K,P 0.003 0.10 0.02 3.0 20.8
7.7
0.57
1.37
1. o
90.0 0.38 0.77 1.3 1.5 25.0 3.6 1.71 1.07 0.08
0.17 0.67
0.014 0.19 0.13 0.105 0.054 1.40 0.007 0.61 0.15 0.15 37.2 41.5
Source of enzyme
Ref.
S . aureua 524 SC S . aureua 147 B. cereua 569/H B. lichenifomis 749/C (wild type) B. lichenifomis 749/C (mutant 19) B. lichenifomis 6346/C (wild type) B. l i c h e n i f m i s 6346/C (mutant 3) E. coli G l l a l E. wli GN 238 E. coli GN 14 E . euli TEM K . aerogenes 418 S . aurew 147 B. coli G l l a l E . coli GN 238 E. coli GN 14 B. cereua 569/H S . auras 524 SC 8.aureus 147
89 78 79 70
70
70
ro 43
44 44
86
90 81
43
44 44
76, 77, m 89 78
~~
V,..I and K,I are the respective constants for the unsubstituted 6-APA; V,,, K,t are the corresponding constants for the indicated 6-APA derivatives. 0
and
brings out the striking differences in the response of the various p-lactamases to a given side chain. Such differences are clearly evident even in closely related enzyme preparations and in some instances (e.g., the mutants of B . Zicheniformis) may result from the replacement of a single amino acid. The role of the side chain in both catalytic and noncatalytic (76) enzyme-substrate interactions has been investigated in considerable detail (reviewed in reference a ) , and a mechanism has been suggested which may explain the rate-controlling effect of side chains on the catalytic activity of p-lactamase (see Section IV,B).
D. STRUCTURAL MODIFICATIONS IN
THE
ENZYME
No data are available on the effect of defined structural modifications on the properties of p-lactamases. Indirect evidence bearing on the
42
NATHAN CITRI
role of the primary structure in catalytic activity comes from the studies of Pollock (23) on the effect of mutations on the properties of the plactamase of B. licheniformis 749/C. It appears that structural mutations, presumably involving a single amino acid replacement in the enzyme molecule, frequently cause a considerable decrease in thermostability (70) along with a decrease in the catalytic activity ( 2 3 ) . The substrate profile is altered as in the case of the mutant strain 749/C/77 (Table IV) with preferential loss of “penicillinase” activity. The change in thermostability and in response to antibodies (see also Section V) implies an altered conformation in such mutant enzymes (23,7 0 ) . A comparison of spontaneously and experimentally released p-lactamase preparations of B . lichenifomis (40) indicates that the ends of the polypeptide chain can vary without affecting the properties of the enzyme. Although such preparations differ in the C- and N-terminus, they are serologically and catalytically indistinguishable (40,63). Similarly, B. lichenifomis 749/C isozymes, separable by starch gel electrophoresis or DEAEchromatography and believed to differ a t the C- or N-terminus C40),have identical substrate specificities (Table V) . Other indirect (genotypic or phenotypic) modifications of the primary structure (70, 91, 92) as well as reversible conformational transitions (52, 67, 93) have been reviewed by Citri and Pollock ( 2 ) and more recently by Pollock ( 4 0 ) .
E. OTHERFACTORS AFFECTING ACTIVITY 1. Effect of pH and Temperature
Typical pH-activity curves obtained with gram-positive p-lactamases and benzylpenicillin show maxima in the range of p H 6.0-7.0 with a rather sharp decline in the alkaline range ( 2 ) . Recent data on gramnegative p-lactamases (25,31,43,44,59,65,90,94-96)show optimal values scattered over the range of pH 5.0-8.5. A shift in the pH-activity curve has been observed when benzylpenicillin was replaced with 6-APA (78,79) or methicillin (54) but not with cephalosporin C ( 3 1 ) . Few data are available on the temperature dependence of p-lactamase activity ( 2 ) . With benzylpenicillin as the substrate, the optimal tem91. 92. 93. 94. 95. 96.
D. A. Dubnau and M. R. Pollock, J . Gen. Microbiol. 41, 7 (1965). M. H. Richmond, BJ 77, 112 (1960). N. Citri and N. Garber, BBA 30, 664 (1958). J. T. Smith and J. M. T. Hamilton-Miller, Nature 197, 976 (1963). J. M. T. Hamilton-Miller, BBRC 13, 43 (1963). G. A . J. Ayliffe, J. Gen. Microbiol. 30, 339 (1!963).
2.
PENICILLINASE AND OTHER
p-LACTAMASES
43
peratures reported range from 30" (44,97)through 35'40" for B. cereus p-lactamases (71,98)to 45"-55" for several enzyme preparations (&,89,98,99).Optimal temperatures for other substrates have not been reported. It has been pointed out (100) that the activation energy of the catalytic reaction (with benzylpenicillin) is generally higher in the gram-posithan in gramtive p-lactamases (7.2-8.8 x 103 cal/mole) (71,100,101) negative enzymes (3.3-5.2 X lo3cal/mole) (31,94,95,100). Replacement of benzylpenicillin with cephalosporin C (31) caused considerable increase in the activation energy. 2. Activators and Inhibitors
As a rule p-lactamase activity does not depend on specific activators or cofactors. The only exception so far is p-lactamase I1 which requires Znz+for stability and activity (37).The metal binding characteristics of this enzyme have been described in a recent study (66).In other cases where the ion content of the assay medium may affect the activity (@,44), no absolute requirements were demonstrated. The search for selective means of inactivation, prompted by clinical considerations, has not been successful, although many substrate analogs act as powerful competitive inhibitors ( 2 ) . On the other hand, irreversible inhibition may result from interaction with substrate analogs which oause labilization of the enzyme (see Section IV,B) . Specific stimulation and inhibition by homologous antibodies is reviewed in Section V. Inhibition of a staphylococcal p-lactamase by certain dipeptides has been reported (102), but it is not clear whether the active site was directly involved. Other remotely related and unrelated compounds as well as metals have been implicated, but the results are largely inconclusive (8,9,20,&). Of the nonspecific inhibitors, the thiol reagents have been most extensively tested and usually found ineffective. This is not surprising in view of the available data on the amino acid composition of p-lactamases (Table 111) which show total absence of cysteine. However, interesting exceptions have been reported. Thiol reagents inhibit the Zn2+-dependent p-lactamase I1 of B . cereus (37, 66) and the &lac97. M. Goldner and R. J. Wilson, Can. J. Microbiol. 7, 45 (1981). 98. E. E. D. Manson, M. R. Pollock, and E. J. Tridgell, J. Gen. Microbiol. 11, 493 (1954). 99. A. Royce, C. Bowler, and G. Sykes, J. Pharm. Pharmacol. 4, 904 (1952). 100. J. T. Smith and J. M . T. Hamilton-Miller, Nature 197, 769 (1963). 101. N. Zyk and N. Citri, BBA 159, 317 (1968). 102. A. K. Saz, D. L. Lowry, and L. J. Jackson, J . Bacten'ol. 82, 298 (1961).
44
NATHAN CITRI
tamases of K . aerogenes and of A . cloacae (65,lOS).I n the case of /3-lactamase I of B. cereus, where the absence of cysteine is reportedly established (Table 111) (lO4),inhibition by PCMB (75)and by Hgz+ ( K , = 1.4 X lo-' M ) (105)has been demonstrated. The former depends on conformative response to substrate analogs (75).The inhibition by Hg2+, which is accompanied by a slight shift in the CD signal in the region of 205-240 nm, is partly reversed by EDTA (105). Inhibition by chelating agents has been reported only in the case of the Zn2+-dependentp-lactamase I1 (47,106). Alcohols may inhibit (107), or stimulate (102).The inhibitory effect of alkyl sulfates (108,109)depends on the length of the alkyl chain (109,110). Thermal inactivation has been studied mainly in the B . cereus enzymes (S8,98,111,llb). The protective effect of certain macromolecules (e.g., gelatin) is observed at loo", but paradoxically not a t 70" (113,114). The effect of various substrates on the thermostability of several p-lactamases (38,77,112,115) has been studied in the context of conformative response (see Section IV,B) . IV. Conformation and Function
A. NONSPECIFIC CONFORMATIONAL TRANSITIONS The absence of disulfide bridges appears to be one of the few characteristics common to all p-lactamases. There are indications that in one case a t least (the cell-bound 7-type p-lactamase of B. cereus) the tertiary 103. J. T. Smith, BJ 87, 40P (1963). 104. M. R. Pollock and M. H. Richmond, Nature 194, 446 (1962). 105. L. D'Souza and R. A. Day, Abstr., 168th A m , Chem. SOC.Meeting, New York p. 60 (1969). 106. L. D. Sabath and E. P. Abraham, Antimicrobial Agents Chemotherapy p. 392 (1966). 107. E. B. McQuarrie and A. J. Lubmann, A B B 5, 307 (1944). 108. R. Brunner, A. Kraushaar, and E. Prohaska, Antibiot. Ann. p. 169 (1960). 109. Z. C . Kaminski, J. Bacteriol. 85, 1182 (1963). 110. E. Rauenbusch, M e d . Chem., Abhandl. Med.-Chem. Forschungsataetterr Farbenfabriken Bayer A . G . 7 , 466 (1963). 111. E. E. D. Manson and M. R . Pollock, J. Gen. Macrobiol. 8, 163 (1953). 112. N. Citri and N. Garber, BBA 67, 64 (1963). 113. D. H. Williams, 111, A. Bondi, A. G . Moat, and F. Ahmad, J. Bacteriol. 91, 257 (1966). 114. For an interpretation of this and related observations, the original report (11.9) should be consulted.
115. N. Zyk and N. Citri, BBA 159, 327 (1968).
2.
PENICILLINASE AND OTHER
P-LACTAMASES
45
structure of the enzyme is nevertheless stabilized by firm, possibly coI n more typical cases, valent, attachment to the cell membrane (38,48). and certainly in all extracellular P-lactamases analyzed so far, the conformation of the enzyme is not constrained by covalent bonds. A likely consequence is the marked flexibility of the conformation of the extracellular p-lactamases of several bacilli (67,77). Reversible transitions in the conformation (and antigenic structure) have been demonstrated in extracellular p-lactamases of B. cereus 569/H (69,67,93) and of B. cereus (52).Thus, a mild treatment with alkali, urea, or guanidine .HCl resulted in a markedly increased levorotation and in exposure of iodine-sensitive regions. Similar changes, including almost total loss of antigenic identity, were observed when the enzyme was adsorbed to charged surfaces. Surprisingly, the adsorbed enzyme was fully active (68)and this led to an inquiry into the possibility that the substrate may restore the conformation of the active site (93,116).
B. SPECIFIC TRANSITIONS : CONFORMATIVE RESPONSE I n a series of papers on the interaction of several purified p-lactamase preparations with various substrates (75,77,101,118,116,117-120), evidence was presented which indicates a possibly meaningful correlation between the structure of the substrate, its effect on the conformation of the enzyme, and the catalytic activity. Much of the evidence (partly reviewed in reference 8) is based on interactions with penicillins differing in the structure of the side chain (substituent R in Fig. 1). As pointed out above (Section 111) the side chain controls the rate of the catalytic reaction. It was subsequently observed that it also controls the conformative response, i.e., the effect of the substrate on the conformation of the enzyme (76).Significantly, the nature of the conformative response could be correlated with the effect on the catalytic activity. Thus, the conformative response to benzylpenicillin and other penicillins with side chains promoting the rate of catalysis is characterized by increased stability of the enzyme molecule. Conversely, penicillins which carry side chains interfering with the catalytic activity labilize the enzyme and facilitate inactivation by heat, urea, proteolysis, iodination, or photooxidation. Such penicillins act as competitive inhibi116. 117. 118. 119. 120.
N. Citri and N. Garber, BBA 38, 50 (1960). N. Garber and N. Citri, BBA 62, 385 (1962). N. Citri and N. Garber, BBRC 4, 143 (1961). N. Citri and N. Garber, J . Pharm. Pharmctcol. 14, 784 (1962) N. Zyk and N. Citri, BBA 146, 219 (1967).
46
NATHAN CITRI
tors of the hydrolysis of benzylpenicillin. The competitive relationship at the catalytic level is reflected in a corresponding shift in the conformative response. These observations led to the conclusion that the side chain of penicillin affects the rate of the reaction by modifying the conformative response of the enzyme. Subsequent studies led to the suggestion that the modified conformative response is strongly reflected in the binding properties of the enzyme (120).Although lacking direct support, this suggestion provides a single explanation for two sets of discrepancies observed when a comparison is made of enzyme-substrate dissociation constants based on the catalytic activity with those based on the conformative response ( 7 4 ) . V. Immunological Studies
The immunology of p-lactamases has been studied fairly extensively (16,23,41,4.3,70,115,121-124) and reviewed elsewhere (70,125).At the molecular level, differences in antigenicity could be usually correlated with differences in composition and physical properties ( 7 0 ) . On the other hand, reversible changes in antigenicity-without apparent loss of activity-could be correlated with reversible conformational transitions ( 5 2 ) . The effect of specific antibodies on the catalytic activity of p-lactamase is usually inhibitory, as expected (125).I n some cases, however, it may be stimulatory ( 7 0 , 1 2 2 ) .This surprising effect depends on the structure of the substrate (101, 122) or on structural mutations in the enzyme ( 7 0 ) . It has been suggested (70,101,115) that the stimulation results from the effect of the antibody on the conformation of the enzyme. Indeed, the effect of antibodies on the conformation of p-lactamase shows a quantitative correlation with their effect on the catalytic activity (101). Specifically, homologous antibodies suppress the conformative response and virtually eliminate the effect of the side chain of the substrate on the rate of catalytic activity (116). These results (cf. Section IV,B) imply a plausible mechanism (115) which may account for both the stimulating and inhibitory effect of antibodies on the activity of p-lactamase. 121. 122. 123. 124. 125.
M. R. Pollock, J. Gen. Microbial. 14, 90 (1956). M. R. Pollock, Immunology 7, 707 (1964). H. K. Rhodes, M. Goldner, and R. J. Wilson, Can. J. Microbiol. 7 , 355 (1961). N. Citri and G. Strejan. Nnture 190, 1010 (1961). M. R. Pollock, Ann. N . Y . Acad. Sci. 103, 989 (1963).
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23 23 25 26 27 27 27 31 35
35 39 40 41 42 44 44 45 46
.
1 Introduction
A . BACKGROUND In 1940 Abraham and Chain ( 1 ) reported that an extract of Escherichia C O Z ~ destroys the antibacterial activity of benzylpenicillin . The authors presented evidence indicating that the extract (and possibly several 1. E . P. Abraham and E . Chain, Nature 146. 837 (1940).
23
24
NATHAN CITRI
other bacterial preparations) contained a penicillin-inactivating enzyme which they named penicillinase. Subsequently, many other bacteria were found to contain or secrete similar enzymes for which the inore general term, p-lactnmnse, Iins I ) c c i i suggesttxl. Tlicrc is 110 autliciiticntcd evidence that organisms other than bacteria produce p-lactamase ( 2 ) . The apparently exclusive and wide distribution of p-lactamascs anioiig diverse bacterial species suggests that this family of enzymes may have cvolvctl in connection with a uniquely bacterial function such as bacterial crll wall formation ( 2 6 ). The role of 1wnicillinasc and other ,8-luctnmascs i n conferring resistance to penicillins a i d the closcly relatctl cephalosporiiis has been amply demonstrated (6-8),niitl rcpcatcdly coiifirmctl in tlic numerous reports tlealing with the clinical aspects of p-lactmiase activity (reviewed in references 6, 7 , 9, 10).At a more fundaincntal lcvc~l,penicillinase has been studied most c.stcnsivcly from the point of view of enzyme induction ( 2 , 11, 1 2 ) , enzyme secretion (13-16), and transfer of genetic elements (17, 18). Earlier investigations a t the niolecular level, which have beeii reviewed in previous editions of this book (.19,20) , have been recently cbspanded to cover additional p-lactamase preparations. The picture which emerges indicates an unexpected diversity of size, structure, and molecular properties. Even more surprising is the variety of catalytic activities, which became increasingly evident as more p-lactamase preparations were iso2. N. Citri and M . R . Pollock. A ~ U U IEnzymol. I. 28, 237 (1966). 3. M. R. Pollock. Brit. M e d . J. IV, 71 (1967). 4. H. G. Boman. K. G. Eriksson-Grennberg, J . Foldes, and E. B. Lindstrom, in “Regulation of Nil[-lric A4cid and Protein Biosynthcsis” (V. V. Koningaherper and I,. 13osch. eds.). Vol. 10. 11. 366. B. B. A . Library, Elsevier Publ. Co., Amstcrdam. 1967. 5. I,. G. Burman, K. Nordstrom. and H. G . Bomnn, J. Bacteriol. 96, 438 (1968). 6. M. Barber, in “The Scientific Basis of Medicine,” Annual Review, 1964, p. 169. Univ. of London, (Athlone), London, 1964. 7. W. M. M. Kirby :ind R. J. Bulger. Ann. 12ee. M e d . 15, 393 (1964). 8. H. C. Neu, BBRC 32, 258 (1968). ‘3. E. Rauenbusch, Antibiot. Cliemolhercipia 14, 95 (1968). 10. J . T. Smith, J . M . T. Hamilton-Miller, nnd It. Knox, J . Phurm. Phurmtrcol. 21, 337 (1969). 11. M. R. Pollock, “The Enzyrncs,” 2nd ed., Vol. 1, p. 619. 1959. 12. M. H. Richmond. Essays Biocliem. 4 , 105 (1968). 13. J . 0. Lxnipen. J . Gen. Microbiol. 48, 249 and 261 (1967). 14. M. R. Pollock. J . Gcn. Microbiol. 26, 239 :ind 267 (1961). 15. D. J . Kushner and M. R . Pollock, J. Gett. Micl-obiol. 26, 255 (1961). 16. D. J . Kushner, J. Gen. Microbiol. 23, 381 (1960). 17. R. P. Novick, Bucteriol. Rev. 33, 210 (1969). 18. M. H. Richmond. A d v n n . Microbiol. Fhysiol. 2, 43 (1968). 19. E. P . Abraham. “The Enzymes,” Vol. 1, Pnrt 2, 1’. 1170, 1951. 20. M . R. Pollock, “The Enzymes,” Vol. 4, p. 269, 1959.
2.
PENICILLINASE AND OTHER
P-LACTAMASES
25
lated and studied against the larger number of scmisyiithetic penicillins and cephalosporins which became available in recent years.
B. DEFINITIONS A N D SPECIFICITY The term p-lactaubase denotes an enzymc which catalyzed thc hydrolysis of the amide bond in the p-lactam ring of 6-amino-penicillanic acid (6-APA) or 7-amino-cephalosporanic acid (7-ACA) and of their iV-acyl derivatives ( 2 ) .Such derivatives arc commonly referred to as penicillins [Fig. 1 (I) 1 and cephalosporins IFig. I (III), (V), and ( V I I ) ] , respectively. There is no evidence that any bond other than thc ainide bond in the intact nucleus of penicillin or cephalosporin is broken by the
(V11)
(VIII)
FIG.1. General structure of substrates tind products of the p-lactamase reaction, Substrates: (I) penicillins; (111). ( V ) , and ( V I I ) rephalosporins. Products: (11) penicilloic acids; ( I V ) , (VI), and (VIII) cephalosporoic acids.
26
NATHAN CITRI
enzyme ( 2 1 ) . Thus the catalytic specificity of p-lactamases appehrs at present to be confined to these two closely related families of antibiotics. The term penicillinuse has been retained in common usage to denote p-lactamase preparations which predominantly catalyze the hydrolysis of penicillins. Conversely, preparations which are more active against cephalosporins have been frequently referred to as cephulosporinases. It is expected that this distinction will disappear as more p-lactamases are found to show a continuous spectrum of activity against penicillins and cephalosporins.
C. OCCURRENCE Although @-lactamaseis widespread among the various bacterial species ( 2 , 9 ) ,the levels of p-lactamase activity within each species are extremely variable. The range (22) and significance of such variations have been the subject of a recent discussion by Pollock ( 2 3 ) . Indeed, studies prompted by variations in the penicillinase-forming ability of bacteria led to important advances in our understanding of the transfer and control of genetic information. In the present context the following observations may be relevant: (1) The gene for p-lactamase is readily lost (or acquired) in strains of bacteria (e.g., staphylococci and enteric bacteria) (24) where it is carried by an extrachromosomal particle (18,2 6 ) . (2) The gene expression (i.e., rate of p-lactamase formation) is often controlled by the mechanism of induction ( 2 , 1 1 , 2 7 ) . (3) A single mutational event, resulting presumably in the replacement of a single amino acid residue, may cause a large decrease in activity (3, 23). 21. The suggestion has been made that certain peptides which have been studied as inducers of p-lactamase in Bacillus cereus and in S. atireus may serve as substrates for the enzyme [A. K. Saz and D. L. Lowery, BBRC 15, 525 (1964); 19, 102 (1965)l. However, there is no evidence that such peptides are in fact hydrolyzed by plactamase. 22. J. M . T . Hamilton-Miller, FEBS Letters 1, 86 (196S). 23. M. R . Pollock, Ann. N . 1’. Acncl. Sci. 151, 502 (1968). 24. In enteric bacteria the p-lactamase gene may be acquired by infection with an R factor (cf. Table IV). This led to the pertinent observation (25) that no plactamase should be regarded as “species-specific” until its gene is shown to bc rhromosomal. 25. N. Datta and M. H. Richmond, BJ 98, 204 (1966). 26. R . P. Novick, Ann. N . Y . Acad. Sci. 128, 165 (1965). 27. M . H . Richmond, Nature 216, 1191 (1967).
2.
PENICILLINASE AND OTHER
P-LACTAMASES
27
D. THECATALYTIC REACIYON By definition, p-lactamase catalyzes the hydrolysis of the P-lactam ring in penicillins or cephalosporins as shown in Fig. 1. The product of the reaction with a penicillin has been identified by Abraham et al. (28) as the corresponding penicilloic acid. Although few other cases have been as rigorously investigated, it is generally accepted that the action of a p-lactamase on a penicillin results in the formation of a relatively stable, single product, penicilloic acid [Fig. 1 (11) 1. An analogous reaction has been postulated for cephalosporins (29,30), but it soon became apparent that the presumed primary product is highly unstable (31, 32) except in the case of cephalosporin y-lactones [Fig. 1 (VII) and (VIII)]. In other cephalosporins the nature of the primary product appears to depend on the substituent R [Fig. 1 (III)] (33).When R’ = H, an unstable “cephttloeporoic acid” is formed [Fig. 1 (V) and ( V I ) ] which is a product analogous to the penicilloic acid. In contrast, when R’ is an acetoxy or pyridinium group, the reaction involves the loss of R’ with the formation of an exocyclic double bond and a rearrangement as shown in Fig. 1 (IV). The resulting compound (Amax = 230 nm) is unstable and undergoes fragmentation (33). II. Molecular Properties
A. PURIFICATION AND PHYSICAL PROPERTIES The numerous reports of p-lactamase activity in various bacterial strains reflect the widespread occurrence and the clinical importance 28. E. P. Abraham, W. Baker, W. R. Boon, C. T. Calam. H. C. Carrington, E. Chain, H. W. Florey, G. G. Freeman, R. Robinson, and H. G. Saunders, in “The Chemistry of Penicillin” (H. T. Clarke, J. R. Johnson, and R. Robinson, eds.). Chaptrr 2. p . 10. Princeton Univ. Press, Princeton, New Jersey, 1949. 29. B. Crompton. M. Jago, K. Crawford, G . G. F. Newton, and E. P. Abraham. BJ 83, 52 (1962). 30. G. G. F. Newton and E. P. Abraham, Nature 175, 548 (1955). 31. L. D. Sabath, M . Jago, and E. P. Abraham, BJ 96, 739 (1965). 32. G. G. F. Newton, E. P. Abraham. and S. Kuwabara, Antimicrobinl Agents
Chemothempy p. 449 (1968). 33. J. M. T. Hamilton-Miller, G . G . F. Newton, and E. P. Abrahani, BJ 116, 371 (1970). 34. Similarly, two distinct p-lactamasee have been observed in pure cultures of Enlerobncter ( 3 6 ) . 35. T. D. Hennessey, J . Gen. Mksobiol. 49, 277 (1967).
TABLE I PURIFIED ~LACTAMASE PREPARATIONS Source of enzyme 1. Bacillus cereus 569/H
extracellular (8-lactamase I) 2. Bacillus cereus 569/H extracellular (8-lactamase 11) 3. Bacillus crrrus 569/H cell bound (8-lactamase 7 ) 4. Bacillus cereus 5/B
ex tracellular
5. Bacillus licheniformis 749/C eutracellular 6. Bacillus licheniformis 749/C cell bound
7. Bacillus lirheniformis 6346/C extracellular and cell bound
Main purification steps 1. Selective adsorption on glass 2. Ammonium sulfate fractionation 1. Ammonium sulfate fractionation 2. Ethanol precipitation 1. Sonic disintegration 2. Selective adsorption on cellulose phosphate 3. 1. CJI-cellulose chromatography Acetone precipitation 2. Ammonium srilfate fractionation 3. 1. Ethanol precipitation Selective adsorption on cellulose phosphate 2. Ammonium sulfate fractionation 1. Lyxozyme and trypsin digestion 2. Ammonium srilfate fractionation 3. DEAIGcelliilose chromatography 1. Ammonium sillfate fractionation
Criteria of homogeneity
Ref.
Electrophoretic and sedimentation patterns Crystallized
36
ChZ-cellulose rechromatography
38
Electrophoretic and sedimentation patterns
39
Sedimentation pattern
40
DEAEcellulose rechromatography
40
Electrophoretic and sediment,ation pat terns
40
37
8. Staphylococcus aurcus (types A, B, and C) extracellular 9. 10.
11. 12. 13. 14.
a
1. Selective adsorption on
cellulose phosphate 2. CM-cellulose chromatography 3. Centrifugation on sucrose gradient 4. Electrophoresis on sucrose gradient 1. Ultrasonic disintegration Escherichia coli TEM (R-TEM)o 2. DEAEcellulose chromatography cell bound 3. Gel filtration Escherichia coli K-12 (Glla1)b 1. Release by spheroplasting 2. SE-cellulose chromatography cell bound 3. Chromatography on hydroxylapatite 1. Osmotic release Escherichia coli DB103 (ATSm)a 2. DEAE-cellulose chromatography cell bound 3. Selective adsorption on hydroxylapatite Escherichia coli W3630 ( R G N Z ~ ~ ) ~1. Ultrasonic disintegration 2. DEAE-cellulose chromatography cell bound 3. CM-cellulose chromatography 1. Ultrasonic disintegration Eschwichia coli W3630 (RGN14). 2. Streptomycin precipitation cell bound 3. DEAE-cellulose chromatography Enterobacter cloacae 214 1. Ultrasonic disintegration cell bound 2. Chromatography on CM-Sephadex G-50 3. Gel filtration
CM-cellulose rechromatography ; sedimentation patterns
DEAE-cellulose rechroma tography
41
25,42
Electrophoretic and sedimentation patterns; immunodiff usion Single band on gel electrophoresis
43
Electrophoretic and sedimentation patterns
44
Electrophoretic and sedimentation patterns
44
Electrophoretic pattern and CM-cellulose rechromatography
45
The 8-lactamase gene was acquired by infection with the R factor shown in parentheses. The 8-lactamase gene is chromosomal.
8
30
NATHAN CITRI
of this group of enzymes. However, in most cases no attempts were made t o isolate the enzyme and the available information is too fragmentary to permit characterization a t the molecular level. Table I lists p-lactamase preparations which have been purified and shown to be homogenous. The different purification procedures, summarized in Table I (25,34-45),reflect to some extent differences in the cellular location and in the physical properties of the various preparations. Of particular interest is the isolation of distinct p-lactamases from a single source such as Bacillus cereus strain 569/H, which yields the three P-lactamase preparations listed in Table I (34).This strain is a constitutive mutant of the inducible strain 569 of B. cereus which produces similar levels of all three variants of p-lactamase upon induction (46-49). The relationship between the cell-bound (7-penicillinase) variant and p-lactamase I (a-penicillinase) has been extensively investigated (38, 46, 50-54) but not clarified. Much less is known about the more recently discovered p-lactamase I1 in relation to the two other varieties. One fascinating question which remains open is the interconvertibility of the three varieties (2,.25,37,38). According to a recent report ( 5 5 ) , p-lactamase I of strain 569 can be further resolved by P-cellulose chromatography. The three fractions thus obtained appear to retain their identity on gel electrophoresis, even in the presence of urea, and yield distinctive tryptic digest maps.
36. M. Kogut, M. R . Pollock, and E. J. Tridgell, BJ 62, 391 (1956). 37. S. Kuwabara, and E. P. Abraham, BJ 103, 27C (1967). 38. N. Citri and A. Kalkstein, ABB 121, 720 (1967). 39. M. R. Pollock, A-M. Torriani, and E. J. Tridgell, BJ 62, 387 (1956). 40. M. R. Pollock, BJ 94, 666 (1965). 41. M. H. Richmond, BJ 94, 584 (1965). 42. N. Datta and P. Kontomichalou, Nature 208, 239 (1965). 43. E. B. Lindstrom, H. G. Boman, and B. B. Steele, J. Bucteriol. 101, 218 (1970). 44. S. Yamagishi, K. O’Hara, T. Sawai, and S. Mitsuhashi, J. Biochem. (Tokvo) 66, 11 (1969). 45. T. D. Hennessey and M. H. Richmond, BJ 109, 469 (1968). 46. M. R. Pollock, J . Gen. Microbid 15, 154 (1956). 47. L. D. Sabath and E. P. Abraham, BJ 98, 11C (1966). 48. R. Sheinin, J. Gen. Microbial. 21, 124 (1959). 49. J. D. Duerksen, BBA 87, 123 (1964). 50. J. D. Duerksen and M. L. O’Connor, BBRC 10, 34 (1963). 51. M. R. Pollock and M. Kramer, BJ 70, 665 (1958). 52. N. Citri, BBA 27, 277 (1958). 53. N. Citri, Bull. Res. Council Israel A9, 28 (1960). 54. E. Ron-Zensiper and N. Citri, Nature 198, 887 (1963). 55. J. Imsande, F. D. Gillin, R . J. Tanis, and A. G. Atherly, JBC 245, 2205 (1970).
2.
PENICILLINASE AND OTHER
p-LACTAMASES
31
Several molecular properties of purified P-lactamase preparations are listed in Table I1 (2,25,36,39,40,41,@, 56-58).I n most cases reported the molecular weight is within the range of 28,000-30,000, which seems to be typical for the gram-positive p-lactamases. Similar values have been reported for some but not all, gram-negative p-lactamases. Thus, the values for the enzymes from several strains of E . coli and Salmonella typhinzurium are in the range of 22,00032,000 (43,44, 59,60).In contrast, lower values (14,00C)-17,000) have been reported for other J3-lactamase preparations isolated from E . coli (Table 11, 26, 4 )and from a strain of Enterobacter cloacae (45). The interesting suggestion has been made (61) that the unusually high molecular activity of extracellular p-lactamases (Table 11) may possibly compensate for the dilution of the secreted enzyme in the growth medium. Partial data on the optical rotation (57)and optical rotatory dispersion (55)of native, denatured, and renatured /3-lactamases of B. cereus suggest fairly strong hydrophobic interactions and compact folding. An a-helix content of 30% has been proposed for the native and renatured enzyme of B. cereus 569 (66).
B. COMPOSITION AND SEQUENCE ANALYSES The amino acid composition of the p-lactamases listed in Table I11
(2, 40,41, 43, 45,54, 55, 58, 62-64) underlines the similarities between
preparations derived from closely related strains of bacteria, especially if the combined basic and acidic amino acid content is compared ( 2 3 ) . The staphylococcal enzymes are remarkably rich in lysine, and the lysine-arginine content of the other gram-positive p-lactamases is almost as high. Complete absence of cysteine appears to be characteristic (2,55), although J3-lactamases which contain a t least one cysteine residue have been reported (37,65,66). 56. J. R. Hall and A. G. Ogston, BJ 62, 401 (1956). 57. N. Citri, N. Garber, and M. Sela, JBC 235, 3454 (1960). 58. M. H. Richmond, BJ 88, 452 (1963). 59. C. Lindqvist and K. Nordstrom, J. Bacterial. 101, 232 (1970). 60. H. C. Neu and E. B. Winshell, ABB, 139, 278 (1970). 61. M. R. Pollock, in “The Bacteria” (I. C. Gunsalus and R. Y . Stanier, eds.), Vol. 4,p. 121. Academic Press, New York, 1962. 62. S.Jacobs, Res. Commun. 16th Congr. Quim. Anal., Lisbon, 1966 p . 212 (1956). 62a.M.H.Richmond, personal communication (1966). 63. R. P.Ambler and R. J. Meadway, Nature 222, 24 (1969). 64. R. J. Meadway, BJ 115, 12P (1969). 65. J. T.Smith, Nature 197, 900 (1963). 66. L. D.Sabath and M. Finland, J. Bacterial. 95, 1513 (1968).
TABLE I1 MOLECULAR PROPERTIES OF PURIFIED &LACTAMASES
Source of enzyme"
B. cereus 569 B. cereus 569/H (1) B. cereus 5/B (4) B. lichenifomis 6346 (7) B. Zicheniformis 749/C (6) B. Zicheniformis 749/C (5) S.nureus A ( 8 ) S. aureus B (8) S . aureus C (8) E. wli TEM (9) E. coli G l h l (10)
Molecular weight 31,500 30,800 35,200 28,000 28,000 28,100 29,600 29,600 29,600 16,700
29,OOO
Molecular activityb 1.60 X 1.53 X 1.48 X 2.10 X 1.18 x 1.08 x 2.0 x 2.8 X 1.9 X 2.0 X 2.08 X
106 1W 106 lW 106 106 104 lo3 l(r 10'
1Oa
Sedimentation coefficient (10'8 X ~ 2 0 2.68e 2.5-2.65" 2.82~ 2.63 2.70 2.66 2.62 2.5 2.5 1.85 3.4
EE
, ~ )
(nm/mg N) 6.0 6.35 7.35 5.65 5.45 4.75 7.38
Electrophoretic mobility0
(cm*/sec/VX
Asc., 1.68; Desc., 1.70d Asc., 1.73; Desc., 1.65d
Desc., 3.4@ Asc., 4.95; Desc., 4.50/ Asc., 4.33; Desc., 3.65/ -
-
13.1
-
~
Numbers in parentheses are the serial numbers of the preparations in Table I. Moles benzylpenicillin hydrolyzed per mole enzyme per minute at 30". The diffusion coefficients determined (66) for preparations 569, 569/H and 5/B are 8.28, 8.38, and 7.80 (107 x D m J . In glycine buffer, p = 0.2, pH 8.4. e In glycine buffer, p = 0.2, pH 8.15. In veronal buffer, p = 0.1, pH 8.15. u Aw., ascending; l)esc., descending. a
b
Ref.
10-6)
36, 66 36, 66, 67 36, 39, 66 2, 40 2, 40 40 68
41 41 26
43
TABLE I11 AMINOACID COMPOSITION OF B-LACTAMASES E. coli E. cloacae Amino acids Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Cysteine WPhPhan N-term AA Total ammonia amide
214 G l l a l D3lo (Ref. 43) (Ref. 46) 9 2 5
10 8
7
12 9 10 15 10 5 6 10 5 3 ? ?
17 4 10 25 18 16
33 19 23 28 17 4 15 23 10 8 0
-
17 4 9 23 18 14 32 20 22 29 17 5 17 24 12 7 0 6 Ala (22)
B. cereus 5/B
B. lichenijormis
569 569/H (Ref. 2, 62)
31 23 6 6 13 11 33 25 9 12 2 4 25 22 6 10 19 21 30 29 17 15 5 1 20 22 17 19 5 7 7 8 0 0 Aspb Lysc
6346/C (Ref. 40)
749/c (Ref. 63, 64)
21 5 10 31 13 5 22 10 19 33 17 1 22 18 4 6 0
19 1 12 28 18 10 24 9 12 19 10 5 9 21 5
24 1 15 37 21 11 27 11 15 26 15
5
-
LYS
7 3 Lysd (20)
-
0 -
5 14 27 6
S. a u r m TypeA TypeB (Ref. 64,68) (Ref. 4 1 ) 43 2 4 39 13 19 18 9 12 18 16 3 19 22 13 7 0 2 LYS (29)
43 2
TypeC (Ref. 4 1 )
42 12 17 22 11 16 21 11 2 15 22 9 7 0 -
45 3 4 39 11 17 18 10 15 19 11 2 18 23 14 7 0 -
(28)
LYS (32)
5
-
The &Iactamase gene was received from G l l a l and both eneymes were purified by identical procedures (Serial No. 11 in Table I). Richmond (@a). c Imsande et al. (66). d See, however, text and Ambler and Meadway (63). a
w
TABLE IV OF VARIOUSSUBSTRATES (RATEOF HYDROLYSIS OF BENZYLPENICILLIN = 100) RELATIVE RATESOF HYDROLYSIS
I@
~
~~
Substrates
Source of enzyme"
Phenoxymethyl- Ampipencillin d i n
Bacillus licheniformis Strain 749/C (3 isozymes) [5] (mutant 77) Strain 6346/C [7] Bacillus cereus Strain 569/H 8-lactamase I [I] plactamase I1 [2] 8-lactamase y [3] Strain 5/B [4] Staphylowccus aureua Types A and B [8] Type C I81 Escherichia w l i Strain TEM (R-TEM) [9] Strain K-12 (Gllal) [lo] Strain W3630 ( h N Z 3 8 ) [I21 Strain W3630 ( h14) ~ [I31 Enlerobacler cloacae 214 [14] a
-
153
64-74
-
-
Oxacillin
Cloxacillin
0.45-0.50 4.0 1.0
-
-
-
4.6-5.0 5.0 13
0.7 89 <0.1 -
40 9
3.5 1.0
10 -
0.5 1.2
-
5 89 5 4
6-APA
120 64 55
-
3.0 89 26 1.8
100 100
-
1.5 0.6
4.5 1.0
50 -
1 -
-
-
110 4 450
-
-
27 292
-
140
-
-
-
130
86
11
-
-
-
-
-
171 96
Cephalosporin C
Methicillin
-
Numbers in brackets are the serial number of the preparation in Table I.
85 1.2 263
Benzylcephalosporin
0.3-0.4 0.9.5-1.06 11.0 15 42
17.5-19.8 275 -
Ref.
23 23 40
3 41 17 -
38, 75 37 38 29, 76
-
-
41,58
0.8 920 -
16 -
65 72 3.6
44
-
-
69
44
1830
8200
45
80
0.02
7200
<0.1 -
Cephaloridine
-
41 25 43
f
ael
E
2.
PENICILLINASE AND OTHER
p-LACTAMASES
35
The data on terminal amino acids (Table 111) support the accepted notion that every p-lactamase consists of a single polypeptide chain. There are no indications to the contrary, although firm evidence is lacking in most cases. Further observations pertaining to the chemical composition of plactamases and comments on the significance of the reported variations can be found in recent reviews ( d , S , 23). Ambler and Meadway have very recently reported on their studies of the amino acid sequence of p-lactamases from representative strains of S. aureus and B. lichenijormis (63, 6 4 ) . The complete sequence of the p-lactamase protein of S. aureus PCI (type A) was determined by the characterization of the peptides produced by digestion with trypsin, chymotrypsin, or pepsin. The sequence analysis of the p-lactamase protein of B . licheniformis 749/C was performed on both the extracellular and the cell-bound enzyme (cf. serial Nos. 5 and 6, Table I ) . The slight differences between the two forms were confined to the N-terminus and attributed to the difference in the release mechanism. Peptides corresponding to about 90% of the molecule have been characterized and their sequences combined to form five larger fragments. The results of these studies are summarized in Fig. 2, which presents the S. aureus sequence and the B. lichenijormis fragments arranged in such a way as to give the best possible match with that sequence. This arrangement implies that the B . licheniformis molecule is several residues longer a t each end (see also Table 111).The authors estimated that the complete sequence of the B. lichenijormis protein will show about 40% identity with that of S. aureus. This estimate has been most recently confirmed (64), and the homology proposed in Fig. 2 is now firmly established. There can be little doubt that both proteins share a common origin and that their divergent evolution accounts for the differences in the catalytic properties listed in Tables IV and V.
111. Catalytic Properties
A. METHODSOF ASSAY The methods most commonly used for the quantitative assay of plactamase are based on one of the following principles: (1) Manometric or alkalimetric titration of the new carboxyl group formed by hydrolysis of the p-lactam ring; (2) iodometric titration of the product, and (3)
1
8
Lys-
Thr-
26 27 -THR- Lss-Thr-
3
4
5
Glu- Met- Lys28
6 7 Lys- Glu-
Asp- Asp-
8 9 10 11 12 13 14 15 Leu- Asn- Asp- LEV- GLU- Lys- Lys- TyrPhe-
Lys-
LD Leu-
Glu-
Glu-
Glu- Phe-
Ser-
29 Gly-
30 Lys-
Glu-
32 VAL-
Gly-
Thr-
Asn- Arg-
Thr-
Val-
Ah-
Tyr-
Arg-
LC Pro- Asp-
Glu-
51 -Ser-
5’2 Ala-
53 Ile-
54 55 56 Leu- Leu- Glu-
5i Gln-
58 Val-
59 Pro-
60 Tyr-
61 62 Asn- Lys-
63 64 65 Leu- Asn- Lys-
Ser-
Ile
(Glx,
GIx,
Asx.
76 -Ala-
79 80 81 87 83 84 85 86 TYI:- Ser- PRO- ILE- Leu- GLT- LYS- Tyr- VAL- GlyLE Tyr- Asn- Pro- IleThr- Glu- Lys- His- Val- Asp-
87
38 89 Asp- Ile-
31
33 34 35 LD- Phe- Am-
Lys-
8
Ah-
-Val-
??
36
Ser-
id
16 17 18 Asn- ALA- His-
19 He-
20 21 GLT- Val-
Ah-
Leu-
Gly-
Asp-
57 38 39 40 41 42 ASP- Lys- ARG- PHE- ALA- Tyr-
Lys-
Arg-
Asx,
Phe- Ala66 Lys-
L e u ) Arg-
-Tyr-
Jer
126
127 -Oh- Leu-
Ile-
(Asp.
Asti) Ala-
128
199 130 G L P - ASP- Lys-
106
Gln-
Asn-
131 13‘7 13) VAL- Thr- Asn-
LCJ- Ile-
112 113 LPS- GluLB Leu- Lys- Gln-
134 Pro-
135 Val-
136 137 AKG- Tyr-
Gly-
Asp-
0111- Val
Asx,
Pro,
Glx)
Arg-
Phe-
151 132 153 .Asp- TIIR- Ser-
154 Thr-
155 Pro-
15G Ala-
157 158 ALA- I’he-
159 Gly-
160 Lys-
161 TI r-
162
Srr)
Ala-
Are
Ala-
Val-
Thr-
Scr-
-Lys-
Asx)
Thr
(Tlir,
(Thr.
Leu-
44 45 46 43 ALA-, S E R - T H R - Ser-
Phe
Ah-
Leu- Asp
48 49 47 LTS- ALA- IleAla-
50 Am-
Leu
Ser-
Thr-
Ilc-
Lp-
68 His-
69 Ile-
70 Asn-
71 Lys-
73 74 ASP- ASP- Ile-
75 Val-
Thr-
Tyr-
Thr-
Arg-
Asp-
Asp-
Leu-
9.5 96 Ile- Glu-
07 Ala-
08
Ser-
99 100 Met- Thr-
67 ValLE Ile-
91 92 93 THR-LEU- LTS- Ala90
94 LEr-
7‘7
Asn-
Thr- Gly- Met- Thr- Leu- Lys
107 10rl 109 110 111 ALA- Asn- A S S - L ~ s - I L E - IleAh-
Ile-
23 24 !25 AbA- LEU- ASP-
Phe- Ala-
Ser!
Leu- Arg.
103 Arg-
124 125 LEV- Lya-
Pro)
Lru-
Glu-
Leu- Arg-
144
145 Ser-
146 Pro-
147 L?s-
148 Stx-
149 Lys-
150 Lys-
(GI?,
Pro,
Thr.
1;1x, Asx,
Asr.
Leu- Ala
118 Lys-
119 Val-
Glu
(Ser,
140 141 142 143 GLU- LEU- Asn- Tyr-
Ile-
138 139 GLU- IleLB Glu- Pro163
Ah,
120 121 122 LJ-s- LTS- Gin-
Glu-
114 115 116 117 ILE- GLP- GLT- Ile-
Lys-
102 103 104 105 -TYR- S E R - Asp- Asn- Thr101
Lys-
Pi! Tyr-
Gly-
Glu-
GI?-
Leu-
1G4 165 166 LEU- A m - Lys- Leu- IleLB Leu- A r e 41a- Phe- A h -
Tyr-
Gln-
Glu-
Val
16T Ah-
168 Asn-
160
Leu-
Glu-
(Asp.
Lys- Lys-
17‘3
174
GI!--
1TO 171 179 LTS- LEU- Ser-
L?-S.
4:
Asp-
LXS- Leo-
Ser-
Glu-
Pro-
175
1.1.- ASU
Lys-
176 - L p
IT7
178
Lys- Plie-
179 180 LEV- Leu-
181 182 ASP- Leu-
NET- Leu- Asn- Asn- Lys- Ser-
183
Leo-
Asp- Trp-
Met-
184
185
186
187
188
1R9 Gly-
190 Asp-
191 Tlir-
192 193 194 LEV- ILE- Lyr-
195 Asp-
196 197 GLT- Val-
Pro-
199 Lys-
200 Asp-
Asn)
Leu- Ile-
Arg-
-4Ia-
Gly
(Asp,
Glu,
Pro.
Trp.
217 .4sn-
919 Val-
220 Ma-
5'11 Phc-
222 Val-
223 224 Tyr- Pro-
225 Lyn-
249
250
193
LB
Lys- Arg- Asp (Thr. Thr,
Leu-
201 202 -Tyr- Lys-
203 204 Val- Ala-
205 206 207 208 209 ASP- LTS- Ser- CLY- Gln-
Gly,
Ala.
Val,
Val)
Asp-
226
227
228
Gln-
Ser-
229 (;hi-
230 231 Pro- Ile-
-lily.
252 ILE- Ser2.51
-1lew
Ile-
-Are Glu-
Ala-
253 254 GLU- Thr-
Glu-
Ah-
255 Ah-
Thr-
Lys- Thr232 Val-
210 211 ALA- IleLB
Ala-
Gly,
212 213 Thr- Tyr-
214 Ala-
215 216 Ser- Arg-
Thr-
(ily-
Ala-
Ala-
Ber-
Tyr-
Cly-
Arg- Asn- Asp- Ilc-
Aln
233 Leu-
234 Val-
235 Ile-
236 Phe-
237 Thr-
238
239 Asn- Lys-
240 241 ASP- Asn-
242 Lys-
244 Asp-
Ala-
Val-
Leu-
Ser-
Ser-
Arg-
Asp
(Ma,
Lys,
256 257 LYR- Srr-
258 259 260 961 VAL- MET- LYS- Glo-
252
263
264
265
266
967
Phc
Lys-
LA Val-
Leu- Asn-
Met- Asn-
Gly-
Val-
218 Asp-
Met-
Lys- Ala-
243 SerLA Asp,
245 246 LTS- Pro-
L?-s) 1 ~ s - Tyr-
247
248
b s n - A>I'- LYS- LEUAsp-
ASP- Lys-
Leu-
Lys
FIG.2. Amino acid sequences of P-lactamases of Staphylococcus aureus PCI (upper line) and of Bacillw licheniformis 749/C (fragments LA to LE, lower line). Matching residues are shown in block letters (upper line). (From Ambler and Meadway, 63.)
TABLE V APPARENTDISSOCIATION CONSTANT^
(pM)
Substrates Source of enzyme" Bacillus licheniformis Strain 749/C (5) Strain 6346/C (7) Bacillus cereus Strain 569 p-lactamase I* Strain 569/H 8-lactamase I (1) p-lactamase I1 (2) Strain 5/B (4) Staphylococcus aureus Type A (8) Type 80/81b Escherichia coli Strain 214 Tb Strain TEM (9) Strain W3630 ( F ~ 238) N (12) Strain W3630 (&N 14) (13) Aerobacter cloacaeb Pseudomanas pyocyanea
Benzylpenicillin 49 9.5
Ampicillin -
-
Methicillin 0.93 0.23
Oxacillin
Cloxacillin
-
-
b
16.6 16.7
Cephalosporin C 50 50
Cephaloridine
Ref.
-
48
110
410
550
-
-
-
-
60 3300 43
1300 3300
-
460 1600 230
570 1600 330
2300 -
3000 -
-
1100 -
3300
-
77, 79, 80 37 20, 77
5 12
170
16000 5000
5000 -
-
120
100 -
-
41, 58, 81
22 5.1 27.8 8 13
35oc 2.3 15.9 34.0 541 -
>46& 22 26.3 222
-
-
-
1000
600 111 400 69 130
44= 3.2
-
-
-
0.lP
1.lC
-
0.g 13.0 16.5c 0.026
Numbers in parentheses are the serial numbers of the preparations in Table I. For purification procedure see original references. c Based on KI with benzylpenicillin as substrate. a
6-APA
-
-
46, 77, 78
78 82, 83
25
44 44
83 31
2.
PENICILLINASE AND OTHER
P-LACTAMASES
39
colorimetric determination of the hydroxaniate formed by residual substrate. The various applications of these and several other principles have been reviewed elsewhere (67, 6‘8).The assay methods, primarily designed for benzylpenicillin, the standard substrate, are generally suitable for other substrates, although in some cases the procedure has to be modified. Specific problems involved in the replacement of benzylpenicillin with other substrates, especially cephalosporins (69) have been recently discussed ( 2 , 31, 32, 7 0 ) . )
B. KINETICSAND SUBSTRATE SPECIFICITY
It is generally agreed that the &I-lactamase reaction is of zero order in the presence of saturating substrate concentration (71) and that the enzyme displays typical Michaelis-Menten kinetics over a wide rangc of concentrations (72). The slight deviations, which have been observed with crude enzyme preparations only (59, 73) deserve further investigation ( 4 ) . Anomalously high dissociation constants recorded with certain substrate combinations (74) have been correlated with the effect of such substrates on the conformation of p-lactamase (see Section IV,B) . Individual “substrate profiles” of variously related p-lactamases and apparent dissociation constants for several substrates are listed in Tables IV (2.9)25, 29,37, 38,40, 41, 43-45, 58, 75, 76) and V (20, 25, 31, 37, 40) 41, &,46,58,76,77--83), respectively. An interesting attempt to correlate )
67. J. M. T. Hamilton-Miller, J. T. Smith, and R. Knox, J. Pharm. Pharmacol. 15,81 (1963). 68. N. Citri, Methods M e d . Res. 10, 221 (1964). 69. E. P. Abraham, Phamtacol. R e v . 14, 473 (1962). 70. M. R. Pollock, J. Fleming, and S. Petrie, Proc. 2nd Meeting Federation Euro-
pean Biochem. SOC.,1966 p. 139. Pergamon Press, Oxford, 1967. 71. R. J. Henry and R. D. Housewright, JBC 167, 559 (1947). 72. J. E. Banfield, Experientia 13, 403 (1957). 73. W.Rothe, Pharmazie 5, 25 (1950). 74. N. Zyk and N. Citri, BBA 151, 306 (1968). 75. N. Citri and N. Zyk, BBA 99, 427 (1965). 76. S. M. Chaikovskaya and T. G. Venkina, Antibiotiki 9, 329 (1964). 77. N. Citri, N. Garber, and A. Kalkstein, BBA 92, 572 (1964). 78. R. H. Depue, A. G. Moat, and A. Bondi, ABB 107, 347 (1964). 79. F. R. Batchelor, J. Cameron-Wood, E. B. Chain, and G. N. Rolinson, Proc. R o y . SOC.B154, 514 (1961). 80. S. Kuwabara and E. P. Abraham, BJ 115, 859 (1969). 81. M. H. Richmond, Brit. M e d . Bull. 21, 260 (1965). 82. J. M. T. Hamilton-Miller and J. T. Smith, Nature 201, 999 (1964). 83. J. M. T. Hamilton-Miller, J. T. Smith, and R.Knox, Nature 208, 235 (1965).
40
NATHAN CITRI
these values in a physiologically meaningful way has been made by Pollock (40), who pointed out that under typical ecological conditions the enzyme functions at unsaturating substrate concentrations. Thus the “physiological efficiency” of a p-lactamase depends on both the V,,,,, and the K , value and may be conveniently expressed as the ratio VlnaJKm. There can be little doubt that this ratio is an important parameter in comparing 8-lactamases in any context dealing with the ecology, evolution, and physiological significance of the enzyme (2,25,40,84).
C. STRUCTURAL MODIFICATIONS IN SUBSTRATES The effect of replacement of the APA nucleus with an ACA nucleus on the susceptibility of the substrate to enzymic hydrolysis depends on the nature of the p-lactamase ( 2 ) . It has been richly illustrated in the case of the p-lactamase of Pseudomonas pyocganea (31)and analyzed in terms of conformative response (see Section IV,B) in a study of a B . cereus p-lactamase (75).I n that study the role of the nucleus was examined in both catalytic and noncatalytic enzyme-substrate interactions. Little has been published on the effect of modifications within each nucleus, presumably because such modifications lead, as a rule, to the loss of antibiotic activity. Conversion of the carboxyl groups in penicillins confers partial resistance to p-lactamase (85-87).I n cephalosporins, replacement of the acetyl group in position 3 of the dihydrothiazine ring [ R in Fig. 1, (111)] by pyridine causes increased susceptibility to hydrolysis by p-lactamases of Pseudomonas pyocyanea (31,88),Enterobacter cloacae (45),E . coli (42,88),and both the extracellular and cell-bound enzymes of B . cereus (38)* The most common modification is the replacement of the N-acyl substituent (“side chain” R in Fig. 1 ) , especially in penicillins. Indeed, the differences in K , and relative V,,, values among the penicillins listed in Tables IV and V all result from the differences in the nature of the side chain. Analysis of such data (Table VI) (25,@, 4,70,76,77-79, 81, 89,901 84. M. R. Pollock, Antimicrobial Agents Chemotherapy p. 292 (1965). 85. D. E. Cooper and S.B. Brinkely, JACS 70, 3966 (1948). 86. N . J. Huang, T. A. Seto, J. M. Weaver, A. R. English, T. J. McBride, and G. M. Schull, Antimicrobial Agents Chemotherapy p. 493 (1964). 87. E. Grunberg and G . Beskid, Antimicrobial Agents Chemotherapy p. 619 (1968). 88. L. D. Ssbath and M. Finland, Ann. N. Y. Acad. Sci. 145, 237 (1967). 89. R. P. Novick, BJ 83, 229 (1962). 90. J. M. T. Hamilton-Miller, BJ 87, 209 (1963).
2.
41
PENICILLINASE AND OTHEB ~-LACTAMASES
TABLE VI EFFECTOF SIDE CHAINON CATALYTIC CONSTANTS OF PENICILLINS Side chain Phenylacetyl-
V,..:: V,..P 5.3 16.7 2.6 20.0 10.0
Phenylglycyl-
Dimethoxybenzoyl-
K,r: K,P 0.003 0.10 0.02 3.0 20.8
7.7
0.57
1.37
1. o
90.0 0.38 0.77 1.3 1.5 25.0 3.6 1.71 1.07 0.08
0.17 0.67
0.014 0.19 0.13 0.105 0.054 1.40 0.007 0.61 0.15 0.15 37.2 41.5
Source of enzyme
Ref.
S . aureua 524 SC S . aureua 147 B. cereua 569/H B. lichenifomis 749/C (wild type) B. lichenifomis 749/C (mutant 19) B. lichenifomis 6346/C (wild type) B. l i c h e n i f m i s 6346/C (mutant 3) E. coli G l l a l E. wli GN 238 E. coli GN 14 E . euli TEM K . aerogenes 418 S . aurew 147 B. coli G l l a l E . coli GN 238 E. coli GN 14 B. cereua 569/H S . auras 524 SC 8.aureus 147
89 78 79 70
70
70
ro 43
44 44
86
90 81
43
44 44
76, 77, m 89 78
~~
V,..I and K,I are the respective constants for the unsubstituted 6-APA; V,,, K,t are the corresponding constants for the indicated 6-APA derivatives. 0
and
brings out the striking differences in the response of the various p-lactamases to a given side chain. Such differences are clearly evident even in closely related enzyme preparations and in some instances (e.g., the mutants of B . Zicheniformis) may result from the replacement of a single amino acid. The role of the side chain in both catalytic and noncatalytic (76) enzyme-substrate interactions has been investigated in considerable detail (reviewed in reference a ) , and a mechanism has been suggested which may explain the rate-controlling effect of side chains on the catalytic activity of p-lactamase (see Section IV,B).
D. STRUCTURAL MODIFICATIONS IN
THE
ENZYME
No data are available on the effect of defined structural modifications on the properties of p-lactamases. Indirect evidence bearing on the
42
NATHAN CITRI
role of the primary structure in catalytic activity comes from the studies of Pollock (23) on the effect of mutations on the properties of the plactamase of B. licheniformis 749/C. It appears that structural mutations, presumably involving a single amino acid replacement in the enzyme molecule, frequently cause a considerable decrease in thermostability (70) along with a decrease in the catalytic activity ( 2 3 ) . The substrate profile is altered as in the case of the mutant strain 749/C/77 (Table IV) with preferential loss of “penicillinase” activity. The change in thermostability and in response to antibodies (see also Section V) implies an altered conformation in such mutant enzymes (23,7 0 ) . A comparison of spontaneously and experimentally released p-lactamase preparations of B . lichenifomis (40) indicates that the ends of the polypeptide chain can vary without affecting the properties of the enzyme. Although such preparations differ in the C- and N-terminus, they are serologically and catalytically indistinguishable (40,63). Similarly, B. lichenifomis 749/C isozymes, separable by starch gel electrophoresis or DEAEchromatography and believed to differ a t the C- or N-terminus C40),have identical substrate specificities (Table V) . Other indirect (genotypic or phenotypic) modifications of the primary structure (70, 91, 92) as well as reversible conformational transitions (52, 67, 93) have been reviewed by Citri and Pollock ( 2 ) and more recently by Pollock ( 4 0 ) .
E. OTHERFACTORS AFFECTING ACTIVITY 1. Effect of pH and Temperature
Typical pH-activity curves obtained with gram-positive p-lactamases and benzylpenicillin show maxima in the range of p H 6.0-7.0 with a rather sharp decline in the alkaline range ( 2 ) . Recent data on gramnegative p-lactamases (25,31,43,44,59,65,90,94-96)show optimal values scattered over the range of pH 5.0-8.5. A shift in the pH-activity curve has been observed when benzylpenicillin was replaced with 6-APA (78,79) or methicillin (54) but not with cephalosporin C ( 3 1 ) . Few data are available on the temperature dependence of p-lactamase activity ( 2 ) . With benzylpenicillin as the substrate, the optimal tem91. 92. 93. 94. 95. 96.
D. A. Dubnau and M. R. Pollock, J . Gen. Microbiol. 41, 7 (1965). M. H. Richmond, BJ 77, 112 (1960). N. Citri and N. Garber, BBA 30, 664 (1958). J. T. Smith and J. M. T. Hamilton-Miller, Nature 197, 976 (1963). J. M. T. Hamilton-Miller, BBRC 13, 43 (1963). G. A . J. Ayliffe, J. Gen. Microbiol. 30, 339 (1!963).
2.
PENICILLINASE AND OTHER
p-LACTAMASES
43
peratures reported range from 30" (44,97)through 35'40" for B. cereus p-lactamases (71,98)to 45"-55" for several enzyme preparations (&,89,98,99).Optimal temperatures for other substrates have not been reported. It has been pointed out (100) that the activation energy of the catalytic reaction (with benzylpenicillin) is generally higher in the gram-posithan in gramtive p-lactamases (7.2-8.8 x 103 cal/mole) (71,100,101) negative enzymes (3.3-5.2 X lo3cal/mole) (31,94,95,100). Replacement of benzylpenicillin with cephalosporin C (31) caused considerable increase in the activation energy. 2. Activators and Inhibitors
As a rule p-lactamase activity does not depend on specific activators or cofactors. The only exception so far is p-lactamase I1 which requires Znz+for stability and activity (37).The metal binding characteristics of this enzyme have been described in a recent study (66).In other cases where the ion content of the assay medium may affect the activity (@,44), no absolute requirements were demonstrated. The search for selective means of inactivation, prompted by clinical considerations, has not been successful, although many substrate analogs act as powerful competitive inhibitors ( 2 ) . On the other hand, irreversible inhibition may result from interaction with substrate analogs which oause labilization of the enzyme (see Section IV,B) . Specific stimulation and inhibition by homologous antibodies is reviewed in Section V. Inhibition of a staphylococcal p-lactamase by certain dipeptides has been reported (102), but it is not clear whether the active site was directly involved. Other remotely related and unrelated compounds as well as metals have been implicated, but the results are largely inconclusive (8,9,20,&). Of the nonspecific inhibitors, the thiol reagents have been most extensively tested and usually found ineffective. This is not surprising in view of the available data on the amino acid composition of p-lactamases (Table 111) which show total absence of cysteine. However, interesting exceptions have been reported. Thiol reagents inhibit the Zn2+-dependent p-lactamase I1 of B . cereus (37, 66) and the &lac97. M. Goldner and R. J. Wilson, Can. J. Microbiol. 7, 45 (1981). 98. E. E. D. Manson, M. R. Pollock, and E. J. Tridgell, J. Gen. Microbiol. 11, 493 (1954). 99. A. Royce, C. Bowler, and G. Sykes, J. Pharm. Pharmacol. 4, 904 (1952). 100. J. T. Smith and J. M . T. Hamilton-Miller, Nature 197, 769 (1963). 101. N. Zyk and N. Citri, BBA 159, 317 (1968). 102. A. K. Saz, D. L. Lowry, and L. J. Jackson, J . Bacten'ol. 82, 298 (1961).
44
NATHAN CITRI
tamases of K . aerogenes and of A . cloacae (65,lOS).I n the case of /3-lactamase I of B. cereus, where the absence of cysteine is reportedly established (Table 111) (lO4),inhibition by PCMB (75)and by Hgz+ ( K , = 1.4 X lo-' M ) (105)has been demonstrated. The former depends on conformative response to substrate analogs (75).The inhibition by Hg2+, which is accompanied by a slight shift in the CD signal in the region of 205-240 nm, is partly reversed by EDTA (105). Inhibition by chelating agents has been reported only in the case of the Zn2+-dependentp-lactamase I1 (47,106). Alcohols may inhibit (107), or stimulate (102).The inhibitory effect of alkyl sulfates (108,109)depends on the length of the alkyl chain (109,110). Thermal inactivation has been studied mainly in the B . cereus enzymes (S8,98,111,llb). The protective effect of certain macromolecules (e.g., gelatin) is observed at loo", but paradoxically not a t 70" (113,114). The effect of various substrates on the thermostability of several p-lactamases (38,77,112,115) has been studied in the context of conformative response (see Section IV,B) . IV. Conformation and Function
A. NONSPECIFIC CONFORMATIONAL TRANSITIONS The absence of disulfide bridges appears to be one of the few characteristics common to all p-lactamases. There are indications that in one case a t least (the cell-bound 7-type p-lactamase of B. cereus) the tertiary 103. J. T. Smith, BJ 87, 40P (1963). 104. M. R. Pollock and M. H. Richmond, Nature 194, 446 (1962). 105. L. D'Souza and R. A. Day, Abstr., 168th A m , Chem. SOC.Meeting, New York p. 60 (1969). 106. L. D. Sabath and E. P. Abraham, Antimicrobial Agents Chemotherapy p. 392 (1966). 107. E. B. McQuarrie and A. J. Lubmann, A B B 5, 307 (1944). 108. R. Brunner, A. Kraushaar, and E. Prohaska, Antibiot. Ann. p. 169 (1960). 109. Z. C . Kaminski, J. Bacteriol. 85, 1182 (1963). 110. E. Rauenbusch, M e d . Chem., Abhandl. Med.-Chem. Forschungsataetterr Farbenfabriken Bayer A . G . 7 , 466 (1963). 111. E. E. D. Manson and M. R . Pollock, J. Gen. Macrobiol. 8, 163 (1953). 112. N. Citri and N. Garber, BBA 67, 64 (1963). 113. D. H. Williams, 111, A. Bondi, A. G . Moat, and F. Ahmad, J. Bacteriol. 91, 257 (1966). 114. For an interpretation of this and related observations, the original report (11.9) should be consulted.
115. N. Zyk and N. Citri, BBA 159, 327 (1968).
2.
PENICILLINASE AND OTHER
P-LACTAMASES
45
structure of the enzyme is nevertheless stabilized by firm, possibly coI n more typical cases, valent, attachment to the cell membrane (38,48). and certainly in all extracellular P-lactamases analyzed so far, the conformation of the enzyme is not constrained by covalent bonds. A likely consequence is the marked flexibility of the conformation of the extracellular p-lactamases of several bacilli (67,77). Reversible transitions in the conformation (and antigenic structure) have been demonstrated in extracellular p-lactamases of B. cereus 569/H (69,67,93) and of B. cereus (52).Thus, a mild treatment with alkali, urea, or guanidine .HCl resulted in a markedly increased levorotation and in exposure of iodine-sensitive regions. Similar changes, including almost total loss of antigenic identity, were observed when the enzyme was adsorbed to charged surfaces. Surprisingly, the adsorbed enzyme was fully active (68)and this led to an inquiry into the possibility that the substrate may restore the conformation of the active site (93,116).
B. SPECIFIC TRANSITIONS : CONFORMATIVE RESPONSE I n a series of papers on the interaction of several purified p-lactamase preparations with various substrates (75,77,101,118,116,117-120), evidence was presented which indicates a possibly meaningful correlation between the structure of the substrate, its effect on the conformation of the enzyme, and the catalytic activity. Much of the evidence (partly reviewed in reference 8) is based on interactions with penicillins differing in the structure of the side chain (substituent R in Fig. 1). As pointed out above (Section 111) the side chain controls the rate of the catalytic reaction. It was subsequently observed that it also controls the conformative response, i.e., the effect of the substrate on the conformation of the enzyme (76).Significantly, the nature of the conformative response could be correlated with the effect on the catalytic activity. Thus, the conformative response to benzylpenicillin and other penicillins with side chains promoting the rate of catalysis is characterized by increased stability of the enzyme molecule. Conversely, penicillins which carry side chains interfering with the catalytic activity labilize the enzyme and facilitate inactivation by heat, urea, proteolysis, iodination, or photooxidation. Such penicillins act as competitive inhibi116. 117. 118. 119. 120.
N. Citri and N. Garber, BBA 38, 50 (1960). N. Garber and N. Citri, BBA 62, 385 (1962). N. Citri and N. Garber, BBRC 4, 143 (1961). N. Citri and N. Garber, J . Pharm. Pharmctcol. 14, 784 (1962) N. Zyk and N. Citri, BBA 146, 219 (1967).
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NATHAN CITRI
tors of the hydrolysis of benzylpenicillin. The competitive relationship at the catalytic level is reflected in a corresponding shift in the conformative response. These observations led to the conclusion that the side chain of penicillin affects the rate of the reaction by modifying the conformative response of the enzyme. Subsequent studies led to the suggestion that the modified conformative response is strongly reflected in the binding properties of the enzyme (120).Although lacking direct support, this suggestion provides a single explanation for two sets of discrepancies observed when a comparison is made of enzyme-substrate dissociation constants based on the catalytic activity with those based on the conformative response ( 7 4 ) . V. Immunological Studies
The immunology of p-lactamases has been studied fairly extensively (16,23,41,4.3,70,115,121-124) and reviewed elsewhere (70,125).At the molecular level, differences in antigenicity could be usually correlated with differences in composition and physical properties ( 7 0 ) . On the other hand, reversible changes in antigenicity-without apparent loss of activity-could be correlated with reversible conformational transitions ( 5 2 ) . The effect of specific antibodies on the catalytic activity of p-lactamase is usually inhibitory, as expected (125).I n some cases, however, it may be stimulatory ( 7 0 , 1 2 2 ) .This surprising effect depends on the structure of the substrate (101, 122) or on structural mutations in the enzyme ( 7 0 ) . It has been suggested (70,101,115) that the stimulation results from the effect of the antibody on the conformation of the enzyme. Indeed, the effect of antibodies on the conformation of p-lactamase shows a quantitative correlation with their effect on the catalytic activity (101). Specifically, homologous antibodies suppress the conformative response and virtually eliminate the effect of the side chain of the substrate on the rate of catalytic activity (116). These results (cf. Section IV,B) imply a plausible mechanism (115) which may account for both the stimulating and inhibitory effect of antibodies on the activity of p-lactamase. 121. 122. 123. 124. 125.
M. R. Pollock, J. Gen. Microbial. 14, 90 (1956). M. R. Pollock, Immunology 7, 707 (1964). H. K. Rhodes, M. Goldner, and R. J. Wilson, Can. J. Microbiol. 7 , 355 (1961). N. Citri and G. Strejan. Nnture 190, 1010 (1961). M. R. Pollock, Ann. N . Y . Acad. Sci. 103, 989 (1963).