Active sites of beta-lactamases. The chromosomal beta-lactamases of Pseudomonas aeruginosa and Escherichia coli.

An acyl-enzyme was isolated from certain chromosomal beta-lactamases and a penicillin. The penicillin was cloxacillin which, although it is a substrate for these enzymes, has such a low kcat. that it functions as an inhibitor. The enzymes were from the mutant of Pseudomonas aeruginosa 18 S that produces the beta-lactamase constitutively [Flett, Curtis & Richmond (1976) J. Bacteriol. 127, 1585-1586; Berks, Redhead & Abraham (1982) J. Gen. Microbiol., in the press] and from Escherichia coli K-12 (the ampC beta-lactamase) [Boman, Nordström & Normak (1974) Ann. N.Y. Acad. Sci. 235, 569-586]. The acyl-enzymes have been degraded to determine the residue labelled, and the sequence around it. The residue labelled is serine. The sequences around the labelled serine in these two beta-lactamases are exceedingly similar. However, the sequences are quite different from those around the active site serine in the beta-lactamases previously studied. There is thus more than one class of serine beta-lactamases.     

Mechanism of inhibition of RTEM-2 beta-lactamase by cephamycins: relative importance of the 7 alpha-methoxy group and the 3' leaving group

Cefoxitin is a poor substrate of many beta-lactamases, including the RTEM-2 enzyme. Fisher and co-workers [Fisher, J., Belasco, J. G., Khosla, S., & Knowles, J. R. (1980) Biochemistry 19, 2895-2901] showed that the reaction between cefoxitin and RTEM-2 beta-lactamase yielded a moderately stable acyl-enzyme whose hydrolysis was rate-determining to turnover at saturation. The present work shows first that the covalently bound substrate in this acyl-enzyme has a 5-exo-methylene-1,3-thiazine structure, i.e., that the good (carbamoyloxy) 3' leaving group of cefoxitin has been eliminated in formation of the acyl-enzyme. Such an elimination has recently been shown in another case to yield an acyl-beta-lactamase inert to hydrolysis [Faraci, W. S., & Pratt, R. F. (1985) Biochemistry 24, 903-910]. Thus the cefoxitin molecule has two potential sources of beta-lactamase resistance, the 7 alpha-methoxy group and the good 3' leaving group. That the latter is important in the present example is shown by the fact that with analogous substrates where no elimination occurs at the enzyme active site, such as 3'-de(carbamoyloxy)cefoxitin and 3'-decarbamoylcefoxitin, no inert acyl-enzyme accumulates. An analysis of the relevant rate constants shows that the 7 alpha-methoxy group weakens noncovalent binding and slows down both acylation and deacylation rates, but with major effect in the acylation rate, while elimination of the 3' leaving group affects deacylation only.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-(phenylacetyl)glycyl-D-aziridine-2-carboxylate, an acyclic amide substrate of beta-lactamases: importance of the shape of the substrate in beta-lactamase evolution

Certain acyclic depsipeptides, but not peptides, are substrates of typical beta-lactamases [Pratt, R.F., & Govardhan, C.P. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1302]. This may reflect either the greater chemical reactivity of depsipeptides (and of beta-lactams, the natural substrates) than peptides or the greater ease of distortion of the depsipeptide (ester) than the peptide (amide) group into a penicillin-like conformation. The latter explanation has been shown to be more likely by employment of a novel beta-lactamase substrate. N-(phenylacetyl)glycyl-D-aziridine-2-carboxylate, which combines a high chemical reactivity with a close to tetrahedral amide nitrogen atom. Although this substrate was better (higher kcat/KM) than a comparable depsipeptide for beta-lactamases, it was poorer than the depsipeptide for the Streptomyces R61 D-alanyl-D-alanine peptidase (which catalyzes specific peptide hydrolysis). It therefore seems likely that one vital feature of the putative evolution of a DD-peptidase into a beta-lactamase would have been modification of the active site to, on one hand, accommodate bicyclic beta-lactams and, on the other, exclude productive binding of planar acyclic amides. Certain serine beta-lactamases and the R61 DD-peptidase also catalyze methanolysis and aminolysis by D-phenylalanine of the N-acylaziridine. The latter reaction, the first amide aminolysis shown to be catalyzed by a beta-lactamase, is a very close analogue of the transpeptidase reaction of DD-peptidases. The methanolysis reaction appeared to proceed by way of the same acyl-enzyme intermediate as formed from depsipeptides possessing the same acyl moiety as the aziridine. The kinetics of methanolysis were employed to determine whether acylation or deacylation was rate limiting to the hydrolysis reaction under saturating substrate concentrations. The kinetics of the aminolysis reaction, catalyzed by the Enterobacter cloacae P99 beta-lactamase, showed the characteristics of, and were interpreted in terms of, a sequential mechanism previously deduced for depsipeptides and this enzyme [Pazhanisamy, S., & Pratt, R. F. (1989) Biochemistry 28, 6875-6882]. This mechanism features two separate binding sites, only one of which is productive. Strikingly, the binding of the N-acylaziridine to the nonproductive site was very tight, such that essentially all hydrolysis at substrate concentrations above 0.1Km proceeded via the ternary complex; this could also be true of penicillins.     

Cryoenzymology of beta-lactamases

The cryoenzymology of several different beta-lactamases has been investigated. Particular attention has been paid to the experimental pitfalls of the technique. These include such factors as false bursts at the start of the reaction, instability of the enzymes during turnover, and Km values so high that little of the enzyme is present as a complex. Many of the difficulties in cryoenzymology stem from the use of organic cryosolvents. A novel "salt" cryosolvent has been tested: ammonium acetate solutions can be used down to about -60 degrees C. The enzymes examined are readily soluble, and stable, in this solvent. Nevertheless, out of 17 beta-lactamase beta-lactam systems, only 4 proved suitable for detailed investigation. In two of these, the hydrolysis of nitrocefin or 7-(thienyl-2-acetamido)-3-[[2-[[4- (dimethylamino)phenyl]azo]pyridinio]-methyl]cephem-4-carboxylic acid (PADAC), by beta-lactamase I from Bacillus cereus, substrate was converted into product at a slow enough rate (at -60 or -55 degrees C, respectively) for it to be possible to do successive scans during the course of the reaction. The spectra were those of substrate and product, and no intermediate was detected. The results argue against the accumulation of intermediate acyl-enzyme. The hydrolysis of PADAC by the P99 beta-lactamase from Enterobacter cloacae again showed spectra characteristic of substrate and product, and there was, moreover, a break in the Arrhenius plot; it is possible that a conformational change is (at least partially) rate-determining. The hydrolysis of dinitrophenylpenicillin by the P99 beta-lactamase did show features suggesting the accumulation of acyl-enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics and mechanism of the serine beta-lactamase catalyzed hydrolysis of depsipeptides

Steady-state kinetic parameters have been determined for the hydrolysis of a series of acyclic depsipeptides (ester analogues of acyl-D-alanyl-D-alanine peptides) catalyzed by representative class C (Enterobacter cloacae P99) and class A (Bacillus cereus I, TEM-2, and Staphylococcus aureus PC1) beta-lactamases. The best of these substrates, and the one most used in this work, was m-[[(phenylacetyl)-glycyl]oxy]benzoic acid, whose rates of cleavage could be followed spectrophotometrically. The P99 enzyme also catalyzed the methanolysis of these substrates in aqueous methanol solutions. Quantitative evaluation of the effects of methanol on the kinetics of the competing hydrolysis and methanolysis reactions, and on the product distribution, supports a reaction mechanism involving an acyl-enzyme intermediate whose formation is rate-determining under conditions of substrate saturation. Consideration of the variation of these kinetic parameters with the structure of the depsipeptides and comparison with the analogous parameters for bicyclic beta-lactam substrates suggest that a variety of substrate binding modes exist on this enzyme. The class A enzymes, B. cereus beta-lactamase I and the TEM-2 beta-lactamase, catalyze depsipeptide and benzylpenicillin hydrolyses but not methanolysis. The acyl-enzyme derived from both types of substrate is thus shielded from external nucleophiles; the shielding is therefore not an effect, direct or indirect, of the thiazolidinyl group in the penicilloyl-enzyme. The class A beta-lactamase of the PC1 plasmid of S. aureus is distinctly different from the above two representatives of that class, in that it does catalyze methanolysis of depsipeptides (but not of benzylpenicillin). The methanolysis kinetics suggest that deacylation is rate-determining at saturation, a conclusion supported by the demonstration of an intermediate during the hydrolysis of m-[[(phenylacetyl)glycyl]oxy]benzoate, subsequent to leaving-group departure. The beta-lactamases have thus been shown to catalyze the hydrolysis of specific depsipeptides with comparable facility to that demonstrated by D-alanyl-D-alanine carboxypeptidase/transpeptidases. The former enzymes, however, differ in being unable to cleave the analogous peptides.     

The pH-dependence of class B and class C beta-lactamases.

The classification by structure allots beta-lactamases to (at present) three classes, A, B and C. The pH-dependence of the kinetic parameters for class B and class C have been determined. They differ from each other and from class A beta-lactamases. The class B enzyme was beta-lactamase II from Bacillus cereus 569/H/9. The plots of kcat against pH for the hydrolysis of benzylpenicillin by Zn(II)-requiring beta-lactamase II and Co(II)-requiring beta-lactamase II were not symmetrical, but those of kcat/Km were. A similar feature was observed for the hydrolysis of both benzylpenicillin and cephalosporin C by a class C beta-lactamase from Pseudomonas aeruginosa. The results have been interpreted by a scheme in which two ionic forms of an intermediate can give product, but do so at differing rates.     

The exocellular beta-lactamase of Streptomyces albus G. Purification, properties and comparison with the exocellular DD-carboxypeptidase.

The exocellular beta-lactamase of Streptomyces albus G has been purified to near protein homogeneity. It consists of one single polypeptide chain of mol.wt. 30 000-31 000, has a rather low isoelectric point (at pH 6.0) and contains less lysine (2.1%) and more half-cystine residues than most beta-lactamases from other Gram-positive bacteria. Penicillins are much better substrates than delta 3-cephalosporins; the catalytic-centre activity of good penicillin substrates is 333-500 s-1. The exocellular, mol.wt. 17 000 DD-carboxypeptidase of S. albus G [previously purified to protein homogeneity; Duez, Frère, Geurts, Ghuysen, Dierickx & Delcambe (1978) Biochem. J. 175, 793-800] behaves as an exceedingly poor beta-lactamase, hydrolysing benzylpenicillin into benzylpenicilloate 5 x 10(-6)-fold less rapidly than does the exocellular beta-lactamase. To all appearances, the beta-lactamase has no bivalent cation requirement whereas, as shown elsewhere [Dideberg, Charlier, Dupont, Vermeire, Frère & Ghuysen (1980) FEBS Lett. 117, 212-214, and Dideberg, Joris, Frère, Ghuysen, Weber, Robaye, Delbrouck & Roelands (1980) FEBS Lett. 117, 215-218], the DD-carboxypeptidase possesses one essential Zn2+ ion per molecule. Peptide 'mapping' and immunological studies suggest that the two Streptomyces enzymes probably have very different structural and mechanistic properties.     

Nucleophilic re-activation of the PC1 beta-lactamase of Staphylococcus aureus and of the DD-peptidase of Streptomyces R61 after their inactivation by cephalosporins and cephamycins.

It has been shown previously [Faraci & Pratt (1985) Biochemistry 24, 903-910; (1986) Biochemistry 25, 2934-2941; (1986) Biochem. J. 238, 309-312] that certain beta-lactam-processing enzymes form inert acyl-enzymes with cephems that possess good leaving groups at the C-3' position. These inert species arise by elimination of the leaving group from the initially formed and more rapidly hydrolysing acyl-enzyme, which has the 'normal' cephalosporoate structure. The present paper shows that a strong nucleophile, thiophenoxide, can catalyse the re-activation of three examples of these inert acyl-enzymes, generated on reaction of cephalothin and cefoxitin with the PC1 beta-lactamase of Staphylococcus aureus and of cephalothin with D-alanyl-D-alanine transpeptidase/carboxypeptidase of Streptomyces R61. In view of the reversibility of the elimination reaction, demonstrated in model systems [Pratt & Faraci (1986) J. Am. Chem. Soc. 108, 5328-5333], this catalysis is proposed to arise through nucleophilic addition to the exo-methylene carbon atom of the inert acyl-enzyme to regenerate a more rapidly hydrolysing normal cephalosporoate. Strong support for this scenario was obtained through comparison of the kinetics of the catalysed re-activation reaction with those of turnover of the relevant 3'-thiophenoxycephems, thiophenoxycephalothin and thiophenoxycefoxitin. The enzymes appear to stabilize the products of the elimination reaction with respect to the normal cephalosporoate, but more strongly to destabilize the transition states. The effects of other nucleophiles, including cysteine, glycine amide and imidazole, on the above enzymes and on other beta-lactamases can be understood in terms of the model reaction kinetics and thermodynamics.     

Mechanism of inhibition of the PC1 beta-lactamase of Staphylococcus aureus by cephalosporins: importance of the 3'-leaving group

The hydrolysis of cephalosporins containing good leaving groups at the 3'-position [those used in this study were the chromogenic cephalosporin PADAC [pyridine-2-azo-4'-(N',N'-dimethylaniline) substituted on cephalosporin], cephaloridine, and cephalothin], catalyzed by the Staphylococcus aureus PC1 beta-lactamase, proceeds in two spectrophotometrically observable phases. The first involves formation of an acyl-enzyme intermediate while the second involves partitioning of this intermediate between two pathways. One path yields the normal cephalosporoate (3) from which the 3'-leaving group is spontaneously eliminated in solution to give the 3-methylenedihydrothiazine 2, while the second involves initial elimination of the 3' substituent, thus yielding a second acyl-enzyme intermediate, which then hydrolyzes to give the same final product as from the first pathway. The second acyl-enzyme is relatively inert to hydrolysis (t1/2 congruent to 10 min at 20 degrees C), and its formation thus leads to transient inhibition of the enzyme. The partition ratio between hydrolysis and elimination at the enzyme active site could be determined either spectrophotometrically from burst experiments or from measurements of residual beta-lactamase activity as a function of cephalosporin concentration. This ratio varied with the leaving group ability of the 3' substituent (acetoxy greater than N,N-dimethylaniline-4-azo-2'-pyridinium greater than pyridinium) in the anticipated fashion. The inert acyl-enzyme intermediate was isolated by exclusion chromatography and shown to contain the cephem nucleus, but not the 3' substituent, covalently bound to the enzyme. As would be expected, PADAC, cephaloridine, and cephalothin yielded the same inert intermediate. Cephalosporins with poor or no 3'-leaving groups, e.g., dansylcephalothin and desacetoxycephalothin, neither displayed the branched pathway nor yielded the long-lived acyl-enzyme.     

Crystal structures of the Bacillus licheniformis BS3 class A beta-lactamase and of the acyl-enzyme adduct formed with cefoxitin

The Bacillus licheniformis BS3 beta-lactamase catalyzes the hydrolysis of the beta-lactam ring of penicillins, cephalosporins, and related compounds. The production of beta-lactamases is the most common and thoroughly studied cause of antibiotic resistance. Although they escape the hydrolytic activity of the prototypical Staphylococcus aureus beta-lactamase, many cephems are good substrates for a large number of beta-lactamases. However, the introduction of a 7alpha-methoxy substituent, as in cefoxitin, extends their antibacterial spectrum to many cephalosporin-resistant Gram-negative bacteria. The 7alpha-methoxy group selectively reduces the hydrolytic action of many beta-lactamases without having a significant effect on the affinity for the target enzymes, the membrane penicillin-binding proteins. We report here the crystallographic structures of the BS3 enzyme and its acyl-enzyme adduct with cefoxitin at 1.7 A resolution. The comparison of the two structures reveals a covalent acyl-enzyme adduct with perturbed active site geometry, involving a different conformation of the omega-loop that bears the essential catalytic Glu166 residue. This deformation is induced by the cefoxitin side chain whose position is constrained by the presence of the alpha-methoxy group. The hydrolytic water molecule is also removed from the active site by the 7beta-carbonyl of the acyl intermediate. In light of the interactions and steric hindrances in the active site of the structure of the BS3-cefoxitin acyl-enzyme adduct, the crucial role of the conserved Asn132 residue is confirmed and a better understanding of the kinetic results emerges.     

Imipenem as substrate and inhibitor of beta-lactamases.

The interaction between imipenem, a carbapenem antibiotic, and two representative beta-lactamases has been studied. The first enzyme was beta-lactamase I, a class-A beta-lactamase from Bacillus cereus; imipenem behaved as a slow substrate (kcat. 6.7 min-1, Km 0.4 mM at 30 degrees C and at pH 7) that reacted by a branched pathway. There was transient formation of an altered species formed in a reversible reaction; this species was probably an acyl-enzyme in a slightly altered, but considerably more labile, conformation. The kinetics of the reaction were investigated by measuring both the concentration of the substrate and the activity of the enzyme, which fell and then rose again more slowly. The second enzyme was the chromosomal class-C beta-lactamase from Pseudomonas aeruginosa; imipenem was a substrate with a low kcat. (0.8 min-1) and a low Km (0.7 microM). Possible implications for the clinical use of imipenem are considered.     

Inhibition of class A and class C beta-lactamases by penems: crystallographic structures of a novel 1,4-thiazepine intermediate

A new beta-lactamase inhibitor, a methylidene penem having a 5,6-dihydro-8H-imidazo[2,1-c][1,4]oxazine heterocyclic substituent at the C6 position with a Z configuration, irreversibly inhibits both class A and class C serine beta-lactamases with IC(50) values of 0.4 and 9.0 nM for TEM-1 and SHV-1 (class A), respectively, and 4.8 nM in AmpC (class C) beta-lactamases. The compound also inhibits irreversibly the class C extended-spectrum GC1 beta-lactamase (IC(50) = 6.2 nM). High-resolution crystallographic structures of a reaction intermediate of (5R)-(6Z)-6-(5,6-dihydro-8H-imidazo[2,1-c][1,4]oxazin-2-ylmethylene)-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-3-carboxylic acid 1 with the SHV-1 beta-lactamase and with the GC1 beta-lactamase have been determined by X-ray diffraction to resolutions of 1.10 and 1.38 A, respectively. The two complexes were refined to crystallographic R-factors (R(free)) of 0.141 (0.186) and 0.138 (0.202), respectively. Cryoquenching of the reaction of 1 with each beta-lactamase crystal produced a common, covalently bound intermediate. After acylation of the serine, a nucleophilic attack by the departing thiolate on the C6' atom yielded a novel seven-membered 1,4-thiazepine ring having R stereochemistry at the new C7 moiety. The orientation of this ring in each complex differs by a 180 degrees rotation about the bond to the acylated serine. The acyl ester bond is stabilized to hydrolysis through resonance stabilization with the dihydrothiazepine ring and by low occupancy or disorder of hydrolytic water molecules. In the class A complex, the buried water molecule on the alpha-face of the ester bond appears to be loosely bound or absent. In the class C complex, a water molecule on the beta-face is disordered and poorly activated for hydrolysis. Here, the acyl intermediate is unable to assist its own hydrolysis, as is thought to occur with many class C substrates.     

The penicillin-binding site in the exocellular DD-carboxypeptidase-transpeptidase of Actinomadura R39.

Heat denaturation and Pronase degradation of the complex previously formed between benzylpenicillin and the exocellular DD-carboxypeptidase-transpeptidase of Actinomadura R39 yields a heptapeptide H-Leu-Pro-Ala-Ser-Asn-Gly-Val-OH, where the benzylpenicilloyl group is ester-linked to the serine residue. This linkage is very labile and its hydrolysis causes the release of benzylpenicilloate. In contrast, the native benzylpenicilloyl-enzyme complex is very stable (half-life 70 h at 37 degrees C) and its breakdown proceeds via fragmentation of the bound benzylpenicilloyl group [Fuad, Frère, Ghuysen, Duez & Iwatsubo (1976) Biochem. J. 155, 623-629].     

Enzyme-induced strain/distortion in the ground-state ES complex in beta-lactamase catalysis revealed by FTIR

Class A beta-lactamases hydrolyze penicillins and other beta-lactams via an acyl-enzyme catalytic mechanism. Ser70 is the active site nucleophile. By constructing the S70A mutant, which is unable to form the acyl-enzyme intermediate, it was possible to make stable ES complexes with various substrates. The stability of such Michaelis complexes permitted acquisition of their infrared spectra. Comparison of the beta-lactam carbonyl stretch frequency (nu(CO)) in the free and enzyme-bound substrate revealed an average decrease of 13 cm(-)(1), indicating substantial strain/distortion of the lactam carbonyl when bound in the ES complex. Interestingly, regardless of the frequency of the C=O stretch in the free substrate, when complexed to Bacillus licheniformis beta-lactamase, the frequency was always 1755 +/- 2 cm(-)(1). This suggests the active site environment induces a similar conformation of the beta-lactam in all substrates when bound to the enzyme. Using deuterium substitution, it was shown that the "oxyanion hole", which involves hydrogen bonding to two backbone amides, is the major source of the enzyme-induced strain/distortion. The very weak catalytic activity of the S70A beta-lactamase suggests enzyme-facilitated hydrolysis due to substrate distortion on binding to the enzyme. Thus the binding of the substrate in the active site induces substantial strain and distortion that contribute significantly to the overall rate enhancement in beta-lactamase catalysis.     

The D-methyl group in beta-lactamase evolution: evidence from the Y221G and GC1 mutants of the class C beta-lactamase of Enterobacter cloacae P99

The beta-lactam antibiotics act through their inhibition of D-alanyl-D-alanine transpeptidases (DD-peptidases) that catalyze the last step of bacterial cell wall synthesis. Bacteria resist beta-lactams by a number of mechanisms, one of the more important of which is the production of beta-lactamases, enzymes that catalyze the hydrolysis of these antibiotics. The serine beta-lactamases are evolutionary descendants of DD-peptidases and retain much of their structure, particularly at the active site. Functionally, beta-lactamases differ from DD-peptidases in being able to catalyze hydrolysis of acyl-enzyme intermediates derived from beta-lactams and being unable to efficiently catalyze acyl transfer reactions of D-alanyl-D-alanine terminating peptides. The class C beta-lactamase of Enterobacter cloacae P99 is closely similar in structure to the DD-peptidase of Streptomyces R61. Previous studies have demonstrated that the evolution of the beta-lactamase, presumably from an ancestral DD-peptidase similar to the R61 enzyme, included structural changes leading to rejection of the D-methyl substituent of the penultimate D-alanine residue of the DD-peptidase substrate. This seems to have been achieved by suitable placement of the side chain of Tyr 221 in the beta-lactamase. We show in this paper that mutation of this residue to Gly 221 produces an enzyme that more readily hydrolyzes and aminolyzes acyclic D-alanyl substrates than glycyl analogues, in contrast to the wild-type beta-lactamase; the mutant is therefore a more efficient DD-peptidase. Molecular modeling showed that the D-alanyl methyl group fits snugly into the space originally occupied by the Tyr 221 side chain and, in doing so, allows the bound substrate to assume a conformation similar to that on the R61 DD-peptidase, which has a hydrophobic pocket for this substituent. Another mutant of the P99 beta-lactamase, the extended spectrum GC1 enzyme, also has space available for a D-alanyl methyl group because of an extended omega loop. In this case, however, no enhancement of activity against D-alanyl substrates with respect to glycyl was observed. Accommodation of the penultimate D-alanyl methyl group is therefore necessary for efficient DD-peptidase activity, but not sufficient.     

Terminal truncations in amp C beta-lactamase from a clinical isolate of Pseudomonas aeruginosa.

AmpC beta-lactamases from strains of Pseudomonas aeruginosa have previously been shown to be heterogeneous with respect to their isoelectric point (pI). In order to elucidate the origin of this heterogeneity enzymes were isolated from a clinical isolate of a multiresistant P. aeruginosa strain and biochemically characterized. The purification was accomplished in four chromatographic steps comprising dye-affinity, size-exclusion, hydrophobic interaction chromatography, and chromatofocusing; this resulted in five forms with pI values of 9.1, 8.7, 8.3, 8.2, and 7.6. When analysed by SDS/PAGE and agarose IEF each separated beta-lactamase appeared to be both size- and charge-homogeneous. The specific activities of the variants were very similar. MS of each isolated beta-lactamase form showed minor differences in molecular mass (range 40.0-40.8 kDa). MS of the beta-lactamase with a pI of 8.2 demonstrated the presence of two subforms. The N-terminal sequences of three of the beta-lactamases were identical to the published sequence [Lodge, J.M. , Minchin, S.D., Piddock, L.J.V. & Busby, J.W. (1990) Biochem. J. 272, 627-631], while two variants were truncated by two amino-acid residues, one of which was acidic. The previously published sequence contains an alanine as the ultimate residue, but two of the beta-lactamases showed a substitution of Ala371 for arginine, whereas in the remaining forms C-terminal truncations by one and three residues were found. Our results indicate that the P. aeruginosa strain does not harbour multiple copies of the ampC gene, but rather that the five beta-lactamase isoforms are products of a single structural gene. The combinations of the identified N- and/or C-terminal truncations explained the multiple pI values of the beta-lactamase isoforms.     

Kinetics of turnover of cefotaxime by the Enterobacter cloacae P99 and GCl beta-lactamases: two free enzyme forms of the P99 beta-lactamase detected by a combination of pre- and post-steady state kinetics

Third-generation cephalosporins bearing oximino side chains are resistant to hydrolysis by class C beta-lactamases such as that from Enterobacter cloacae P99. For example, steady state parameters for hydrolysis of cefotaxime by this enzyme are as follows: k(cat) = 0.41 s(-1), K(m) = 17.2 microM, and k(cat)/K(m) = 2.3 x 10(4) s(-1) M(-1). On the other hand, however, the K(i) value for cefotaxime as an inhibitor of cephalothin hydrolysis is 27 nM. The discrepancy between K(m) and K(i) indicated that a real steady state had not been achieved in at least one of these experiments. Analysis indicated that only two to three cefotaxime turnovers occurred during the K(i) determination. This suggested that the first few turnovers of cefotaxime by the P99 beta-lactamase may be different from those in the subsequent steady state. A direct pre-steady state experiment confirmed this hypothesis. The simplest reaction scheme that fitted the data involved replacement of the initial enzyme form, E, which bound cefotaxime tightly, with a second more weakly binding form, E', by partitioning of the acyl-enzyme intermediate during the first few turnovers. Steady state turnover of cefotaxime then largely involved E' as the free enzyme form. E' slowly reverted to E in the post-steady state regime. Further evidence for this scheme included quantitative analysis of the post-steady state and observation of a difference in the catalytic activity of E and E' in 2 M ammonium sulfate. The kinetics of P99 beta-lactamase-catalyzed hydrolysis of an acyclic depsipeptide substrate bearing a third-generation cephalosporin side chain showed that the side chain is necessary but not sufficient for production of resistance to beta-lactamase; a combination of the side chain and the dihydrothiazine ring of a cephalosporin is required. The beta-lactamase of E. cloacae GC1, an extended spectrum mutant of the P99 enzyme, rapidly hydrolyzes third-generation cephalosporins, without the structural transition described above. The flexibility of the extended Omega loop of the GC1 enzyme probably leads to this situation. Conformational restriction of the loop in the P99 enzyme is probably responsible for the long-lived acyl-enzyme intermediate and the transition to E' induced by cefotaxime.     

Why clinically used tazobactam and sulbactam are poor inhibitors of OXA-10 beta-lactamase: Raman crystallographic evidence

The clinically used inhibitors tazobactam and sulbactam are effective in the inhibition of activity of class A beta-lactamases, but not for class D beta-lactamases. The two inhibitors exhibit a complex multistep profile for their chemistry of inhibition with class A beta-lactamases. To compare the inhibition profiles for class A and D enzymes, the reactions were investigated within OXA-10 beta-lactamase (a class D enzyme) crystals using a Raman microscope. The favored reaction pathway appears to be distinctly different from that for class A beta-lactamases. In contrast to the case of class A enzymes that favor the formation of a key enamine species, the OXA-10 enzyme forms an alpha,beta-unsaturated acrylate (acid or ester). Quantum mechanical calculations support the likely product as the adduct of Ser115 to the acrylate. Few enamine-like species are formed by sulbactam or tazobactam with this enzyme. Taken together, our results show that the facile conversion of the initial imine, formed upon acylation of the active site Ser67, to the cis- and/or trans-enamine is disfavored. Instead, there is a significant population of the imine that could either experience cross-linking to a second nucleophile (e.g., Ser115) or give rise to the alpha,beta-unsaturated product and permanent inhibition. Alternatively, the imine can undergo hydrolysis to regenerate the catalytically active OXA-10 enzyme. This last process is the dominant one for class D beta-lactamases since the enzyme is not effectively inhibited. In contrast to sulbactam and tazobactam, the reactions between oxacillin or 6alpha-hydroxyisopropylpenicillinate (both substrates) and OXA-10 beta-lactamase appear much less complex. These compounds lead to a single acyl-enzyme species, the presence of which was confirmed by Raman and MALDI-TOF experiments.     

Spectral observation of an acyl-enzyme intermediate of lipoprotein lipase

There have been several studies indicating that hydrolysis reactions of fatty acid esters catalyzed by lipases proceed through an acyl-enzyme intermediate typical of serine proteases. In particular, one careful kinetic study with the physiologically important enzyme lipoprotein lipase (LPL) is consistent with rate-limiting deacylation of such an intermediate. To observe the spectrum of acyl-enzyme and study the mechanism of LPL-catalyzed hydrolysis of substrate, we have used a variety of furylacryloyl substrates including 1,2-dipalmitoyl-3-[(beta-2-furylacryloyl)triacyl]glyceride (DPFATG) to study the intermediates formed during the hydrolysis reaction catalyzed by the enzyme. After isolation and characterization of the molecular weight of adipose LPL, we determined its extinction coefficient at 280 nm to quantitate the formation of any acyl-enzyme intermediate formed during substrate hydrolysis. We observed an intermediate at low pH during the enzyme-catalyzed hydrolysis of (furylacryloyl)imidazole. This intermediate builds early in the reaction when a substantial amount of substrate has hydrolyzed but no product, furylacrylate, has been formed. The acyl-enzyme has a lambda max = 305 nm and a molar extinction coefficient of 22,600 M-1 cm-1; these parameters are similar to those for furylacryloyl esters including the serine ester. These data provide the first spectral evidence for a serine acyl-enzyme in lipase-catalyzed reactions. The LPL hydrolysis reaction is base catalyzed, exhibiting two pKa values; the more acidic of these is 6.5, consistent with base catalysis by histidine. The biphasic rates for substrate disappearance or product appearance and the absence of leaving group effect indicate that deacylation of intermediate is rate limiting.     

Structures of ceftazidime and its transition-state analogue in complex with AmpC beta-lactamase: implications for resistance mutations and inhibitor design

Third-generation cephalosporins are widely used beta-lactam antibiotics that resist hydrolysis by beta-lactamases. Recently, mutant beta-lactamases that rapidly inactivate these drugs have emerged. To investigate why third-generation cephalosporins are relatively stable to wild-type class C beta-lactamases and how mutant enzymes might overcome this, the structures of the class C beta-lactamase AmpC in complex with the third-generation cephalosporin ceftazidime and with a transition-state analogue of ceftazidime were determined by X-ray crystallography to 2.0 and 2.3 A resolution, respectively. Comparison of the acyl-enzyme structures of ceftazidime and loracarbef, a beta-lactam substrate, reveals that the conformation of ceftazidime in the active site differs from that of substrates. Comparison of the structures of the acyl-enzyme intermediate and the transition-state analogue suggests that ceftazidime blocks formation of the tetrahedral transition state, explaining why it is an inhibitor of AmpC. Ceftazidime cannot adopt a conformation competent for catalysis due to steric clashes that would occur with conserved residues Val211 and Tyr221. The X-ray crystal structure of the mutant beta-lactamase GC1, which has improved activity against third-generation cephalosporins, suggests that a tandem tripeptide insertion in the Omega loop, which contains Val211, has caused a shift of this residue and also of Tyr221 that would allow ceftazidime and other third-generation cephalosporins to adopt a more catalytically competent conformation. These structural differences may explain the extended spectrum activity of GC1 against this class of cephalosporins. In addition, the complexed structure of the transition-state analogue inhibitor (K(i) 20 nM) with AmpC reveals potential opportunities for further inhibitor design.