Biochem. J. (1982) 203, 223-234 223 Printed in Great Britain

A2- and A3-, penicillinate and 6-unsubstituted

Intrinsic reactivity and interaction with P-lactamases and D-alanyl-D-alanine-cleaving serine peptidases

Jean-Marie FRERE, Judith A. KELLY,* Daniel KLEIN and Jean-Marie GHUYSEN Service de Microbiologie, Faculte de Medecine, Institut de Chimie, B6, Universite de Liege, Sart-Tilman, B-4000 Liege, Belgium and Paul CLAES and Hubert VANDERHAEGHE Rega Instituut, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium

(Received 5 October 1981/Accepted 26 November 1981) The intrinsic reactivity of A2- and A3-deacetoxy-7-phenylacetamidocephalosporanates, penicillanate, unsubstituted, 2-methyl- and 2-phenyl-penems and other ,J-lactam has been expressed in terms of the second-order rate constant (M-1 * s-'(OH-)) for the hydrolysis of the /3-lactam amide bond by OH- at 37°C. The values thus obtained have been compared with the second-order rate constants (M-1 * s-'(enzyme)) for the opening of the same ,J-lactam amide bond during interaction with the fJ-lactamases of Streptomyces albus G and Actinomadura R39 and the D-alanyl-D-alanine-cleaving serine peptidases of Streptomyces R61 and Actinomadura R39. Depending on the cases, the accelerating effect due to enzyme action and expressed by the ratio m-Is-I(enzyme)/M-*Is-I(OH-) varies from less than 2 to more than 1 x 106. The primary parameter that governs enzyme action is the goodness of fit of the fJ-lactam molecule to the enzyme cavity rather than its intrinsic reactivity. With the D-alanyl-D- alanine-cleaving serine peptidases, the three penems studied form intermediate complexes characterized by very short half lives of 14-100s, values significantly lower than those exhibited by most fl-lactam compounds.

16-Lactam compounds with widely varying bi- immobilize the enzymes, at least for some time, in cyclic fused-ring systems and substituents have been the form of serine ester-linked penicilloyl (cephalo- synthesized or isolated from natural sources (Hamil- sporoyl)-enzyme complexes. To answer the ques- ton-Miller & Smith, 1979; Gregory, 1981; Salton & tion, two fl-lactamases and two D-alanyl-D-ala- Shockman, 1981). Modifications of the pyramidal nine-cleaving serine peptidases were selected. The character of the f,-lactam nitrogen (Woodward, fJ-lactamases were those secreted by Streptomyces 1980) and the presence of side-chains facilitating albus G (the 'G ff-lactamase'; Deuz et al.. 1981a) electron delocalization outside the JJ-lactam ring and Actinomadura R39 (the 'R39 ,B-lactamase'; (Boyd & Lunn, 1979) are structural variations that Deuz et al., 1982). The D-alanyl-D-alanine-cleav- affect the intrinsic reactivity. The question therefore ing serine peptidases were those secreted by Strepto- arises to what extent this intrinsic reactivity (in the myces R61 (the 'R61 peptidase'; Frere et al., 1973, ground-state conformation) influences the reactions 1976) and the same Actinomadura R39 as above of the fl-lactam compounds with fl-lactamases (the 'R39 peptidase'; Frere et al., 1974; Duez et al., and D-alanyl-D-alanine-cleaving serine peptidases. 1981b). In these and other (Kelly et al., 1981a,b) 'Classical' (cephalosporins) are sub- studies, the interactions had been described between strates of the JJ-lactamases; they are hydrolysed into the four selected enzymes and a large number of biologically inactive penicilloate (cephalosporoate) compounds belonging to various f,-lactam families. derivatives. 'Classical' penicillins (cephalosporins) To cover a wider range of intrinsic reactivity, the are mechanism-based inactivators of the serine 2- A2-deacetoxy-7-phenylacetamidocephalo- D-alanyl-D-alanine-cleaving serine peptidases; they sporanate, its 3-cephem analogue, the 6-unsubsti- tuted penicillanate and three 6-unsubstituted * Permanent address: Institute of Materials Science, penems have been investigated. The results thus The University of Connecticut, Storrs, CN 06268, U.S.A. obtained are described in the present report. Vol. 203 0306-3275/82/040223-1 2$01.50/1 (© 1982 The Biochemical Society 224 J.-M. Frere and others

Materials and methods of 1 M-sodium acetate buffer, pH 4.0, and the Enzymes and buffers amounts of hydrolysed products were estimated with the starch/I2 procedure as described by Johnson et The G and R39 ,-lactamases and the R61 and al. (1975). With the three penems, the determina- R39 peptidases were purified as previously de- tions were made on 300,1 samples and the hydro- scribed (Duez et al., 198 la, 1982; Frere et al., 1973, lysis was followed by spectrophotometric measure- 1974). Estimation of enzyme activity and, in the case ments with a Beckman recording DU-8 spectro- of the peptidases, enzyme inactivation, were carried photometer at the appropriate wavelengths (Table out at 300C for the JJ-lactamases and at 37°C for 1). In all cases, the fl-lactam compound con- the peptidases in the following buffers (unless centrations were about the same as the Km values. otherwise stated): 5mM-sodium phosphate, pH7.0 (R61 peptidase); 0.1 M-Tris/HCI, pH 8.0, con- Interaction between D-alanyl-D-alanine-cleaving ser- taining 0.1 M-NaCl and 50mM-MgCl2 (R39 pep- inepeptidases and /3-lactam compounds tidase); and 50mM-sodium phosphate, pH 7.0 (the G 'Classical' penicillins and A3-cephalosporins in- and R39 ,B-lactamases). The stock solution of the G activate the D-alanyl-D-alanine-cleaving serine pep- fJ-lactamase (and the dilutions made from it) was in tidases according to the model: 50mM-sodium phosphate buffer, pH 7.0, containing 10% (v/v) glycerol and 10% (v/v) ethylene glycol. K kE2 k These polyols were not included in the reaction mixtures for the determination of enzyme activity. where E=enzyme, I =fl-lactam compound, El = fi-Lactam compounds Michaelis complex, EI* = acyl-enzyme inter- A3 - 7/f - Phenylacetamidodeacetoxycephalospor - mediate, P(s) = degradation product(s), K = disso- anic acid was prepared from S- ciation constant and k+2 and k+, = first-order rate sulphoxide (de Koning et al., 1975). After esteri- constants (Frere et al., 1975a,b). The higher the fication with diazomethane, this compound was k+2/K value and the lower the k+3 value, the better transformed into A2-7/f-phenylacetamidodeacetoxy- the fl-lactam as enzyme inactivator. The cephalosporanic acid, using the method described first step of the interaction is assumed to be a for the phenoxyacetamido derivative (Van Hey- quasi-equilibrium process. No evidence to the ningen & Ahern, 1968): a (p.p.m.) (60 MHz, in contrary has been found by Frere et al. (1975a) and [2Hlchloroform/[2Hldimethyl sulphoxide, with tetra- Fuad et al. (1976). Moreover, the value of the methylsilane as internal standard) 1.9 (broad s, second-order rate constant (k+2/K) is always sub- CH3), 3.6 (s, C6H5-CH2), 4.68 (broad s, H-4), 5.25 stantially smaller than 107M-1.s-1, which, as dis- (d, J= 4Hz, H-6), 5.55 (double d, J= 4 and 8Hz, cussed by Brocklehurst (1979), is the probable lower H-7), 5.90 (broad s, H-2), 7.30 (s, C6HA), 7.85 (d, limit of k+ 1. J = 8Hz, NH). T.l.c. on pre-coated silica gel F254 In the cases of penicillanate and both R61 and plates with benzene/acetone/acetic acid (60:39:1, R39 peptidases, of A3-deacetoxy-7-phenylacet- by vol.) gave: A2-cephem, RF 0.38; A3-cephem, RF amidocephalosporanate and both R61 and R39 0.46. Penicillanic acid was a gift from Pfizer Central peptidases, and of A2-deacetoxy-7-phenylacet- Research, Sandwich, Kent, U.K. The unsubstituted amidocephalosporanate and the R39 peptidase, the , 2-methylpenem and 2-phenylpenem [racemic k+2/K value (for enzyme inactivation) and the k+3 mixtures of SR(+)- and 5S(-)-isomers] were gifts value (for enzyme reactivation) were estimated as from Dr. J. Gosteli, formerly of the Woodward described by Kelly et al. (198 lb). To determine the Institute, Basel, Switzerland, whose present address k+3 value, the excess of the ,8-lactam compound used is Cerecon, Badendorf B.L., Switzerland. The to inactivate the peptidases had to be destroyed with structure of these six compounds is shown in Table a sufficient amount of fl-lactamase; the R39 ,6- 2. For other 6-lactam compounds, see Kelly et al. lactamase was used for this purpose. The rate of (1981a,b). inactivation of the R39 peptidase (ka = k+2/{ 1 + (K! [fl-lactam])}, according to the model) was pro- Interaction between f-lactamases and filactam portional to [f8-lactaml up to 4pM (3-cephem), 13,UM compounds (2-cephem) and 70UM (penicillanate). At higher The six fJ-lactam compounds under study were concentrations, the inactivation was too rapid to be substrates of the fJ-lactamases and the kinetic followed accurately. The rate of inactivation of the parameters Km and Vmax were calculated from R61 peptidase by the 3-cephem was proportional to Lineweaver-Burk plots. With the penicillanate and [f,-lactaml up to 10,UM. With penicillanate, the plot A2- and A3-deacetoxy-7-phenylacetamidocephalo- of ka versus [,6-lactaml showed deviation from sporanates, the determinations were made on 30p1 linearity permitting evaluation of the K and k+2 samples. The reaction was stopped by adding 0.2ml constants. 1982 ,B-Lactamases and D-alanyl-D-alanine-cleaving serine peptidases 225

The interaction between A2-deacetoxy-7-phenyl- values were estimated (assuming a competitive acetamidocephalosporanate and the R61 peptidase model) from the slope of the line v/v; versus proceeded through an intermediate EI*, which was [fl-lactaml where v and vi are the rates of D-alanine too short-lived to permit evaluation of the k+3 and release in the absence and in the presence of various k+2/K values by the reference procedures. (i) To penem concentrations. This latter procedure, when determine the k+3 value, the technique used was that applied to the interaction between the unsubstituted previously described (Kelly et al., 1981b) for penem and the R61 peptidase, yielded a K1 (or Ki) measuring the rate of degradation of the complex value of 70,M; this value was in excellent agree- formed between the R39 peptidase and 6-amino- ment with the 80,UM value obtained by the direct penicillanate (and using the R39 /1-lactamase to procedure. (iii) Finally, in another series of experi- rapidly destroy the excess of the 2-cephem). (ii) To ments, the procedure described above to determine determine the k+2/K value, the procedure made use the k+3 value for the interaction between the R61 of measurements of the residual peptidase activity at peptidase and A2-deacetoxy-7-phenylacetamido- the steady state (ASS) in the presence of 1.5mM- cephalosporanate was also applied to the inter- diacetyl-L-Lys-D-Ala-D-Ala [this concentration was action between the R39 peptidase and the three well below the Km value (12 mM), so that the addition penems. Samples of the R39 peptidase (1,ug) were of the tripeptide had little effect on the ASS valuel. incubated with saturating concentrations (0.1 mM) of Under these conditions, the penems at 370 C in a total volume of 200,u1 ofthe Aok+3 Hepes/NaCl/MgCl2 buffer (see above). The reac- tion mixtures were then supplemented with 400nmol sska + k+3 of diacetyl-L-Lys-D-Ala-D-Ala and 10,ug of G where AO= enzyme activity in the absence of the f-lactamase (a quantity sufficient to destroy the 2-cephem. The rate of enzyme inactivation, ka was excess of the penems in less than 1 s) and the proportional to [2-cephemi for three [2-cephemi amounts of released D-alanine were monitored as a values ranging between 20 and 60pM and yielded a function of time as indicated above. The k+3 values k+2/K value of 75M-l-s1. At the same 2-cephem thus obtained were close to the kcat. values. concentrations, a 60M-1 -s1 value for the k+2/K ratio was calculated from the decrease ofthe enzyme activity during the first 15 min of contact, before the Susceptibility of the f/-lactam amide bond to hydro- steady state was established. An average value of lysis by OH- at 37 °C andpH12.0 68 M-1 s-'s was therefore adopted. The susceptibility to attack by OH- of the With the three penems and both R61 and R39 ,-lactam amide bond of the six ,B-lactam com- peptidases, the steady state was so rapidly estab- pounds under study was investigated in 1 M-K2HPO4 lished that it was impossible to determine the rates of adjusted to pH 12.0 with 10M-KOH. The results enzyme inactivation. These compounds were then were expressed in terms of second-order rate regarded as substrates. With the R61 peptidase, the constants (M-1 * s-') for hydrolysis. The reaction was kinetic parameters Km and Vmax (at 370C) were shown to be first order in OH- between pH 11 and estimated on the basis of Lineweaver-Burk plots by 12 with C and cephalothin (and was spectrometric measurements at the appropriate assumed to be so in the other cases). A slight wavelengths (Table 1). With the R39 peptidase, the decrease in the reaction rate was observed by Km values were too small to be determined accur- decreasing the concentration of K2HPO4 from 1.0 to ately by this direct procedure; consequently, they 0.1 M, but the effect was negligible when compared were measured by substrate competition experi- with the first-order dependency on OH-. The same ments under the following conditions. (i) With the study was extended to 19 other fJ-lactam com- 2-methylpenem, samples containing the R39 pep- pounds (belonging mainly to the 3-cephem and tidase (0.2,ug), four concentrations of diacetyl-L- penam families) whose interactions with the G and Lys-D-Ala-D-Ala (from 0.8 to 3.2mM) and various R39 ,-lactamases and the R61 and R39 peptidases concentrations of 2-methylpenem (from 0 to 22,uM) had been previously investigated (see the Intro- were incubated for 10min at 370C in 40,l (total duction). Two procedures were used. With the volume) of 100mM-Hepes/HCI buffer, pH8.0, con- iodometric procedure (which was applied to all the taining 0.1 M-NaCl at 50mM-MgCI2 and the compounds except , nitrocefin and amounts of released D-alanine were estimated under ), the initial concentration of the /3- initial-rate conditions. The inhibition pattern was lactam compounds was 1 mm. Samples (5-10l1) clearly competitive and-yielded, for this particular were removed after increasing times of incubation at penem, a K1 value (which, in fact, is its Km value) of 370C, supplemented with 0.2 ml of 1 M-sodium 1.5 UM. (ii) With the unsubstituted penem and the acetate buffer, pH4.0, (to stop the reaction) and the 2-phenylpenem, a fixed diacetyl-L-Lys-D-Ala-D-Ala amounts of hydrolysed product were estimated by concentration (2 mM) was used and the K, (i.e. Km.) the starch/I2 procedure (Johnson et al., 1975). With Vol. 203 226 J.-M. Frere and others

Table 1. Variations of the molar absorption coefficients off/-lactam compounds caused by the hydrolysis of the fi-lactam amide bond by either 13-lactamase action or treatment with OH- fl-Lactam compound Ac (M- I cm-') Wavelength (nm) Benzylpenicillin -570 240 A3-Deacetoxy-7-phenylacetamidocephalosporanate -2460 258 Cephalothin -7200 260 Cephaloglycine -5700 260 Cephaloridine -10000 260 A2-Deacetoxy-7-phenylacetamidocephalosporanate +5200 280 N-Formimidoylthienamycin -7400 298 2-Methylpenem -3900 298 Unsubstituted penem -5000 305 2-Phenylpenem -4000 318 Nitrocefin -10000 386

the spectrophotometric procedure (which was ap- Interaction ofthe 2-cephem (compound 1), 3-cephem plied to the compounds listed in Table 1), the initial (compound 2), penicillanate (compound 11) and the concentrations of the fJ-lactam compounds ranged penems (compounds 22-24) with the R39 and G from 0.1-0.5mM and the total volume of the f/-lactamases and the R61 and R39 D-alanyl-D- solutions was 300,ul. The absorbance variations of alanine-cleaving serine peptidases the solutions were recorded as a function of time at the appropriate wavelengths (Table 1). The kinetic parameters for the interaction between the various fl-lactam compounds under considera- tion and the R39 and G f-lactamases are given in Non-planarity of the fi-lactam nitrogen of the Table 3, and for the R39 and R61 peptidases are fi-lactam compounds given in Tables 4 and 5. Note that each of the three Non-planarity of the fl-lactam nitrogen was penems studied was a racemic mixture of 5R(+)- expressed by the distance (h) between the apex and and 5S(-)-isomers. As shown in Fig. 1, only one the base of a trigonal'pyramid, where the nitrogen isomer [most likely the SR(+)-isomerl was sensitive atom is at the apex and its three substituents are at to the fl-lactamases. Indeed, a prolonged incubation the corners of the base (Sweet & Dahl, 1970; of the penems with the fl-lactamases resulted only in Woodward, 1980). The h values for the unsub- a partial disappearance of the absorption band in the stituted, 2-methyl- and 2-phenyl-penems were those near-u.v. (contrary to what was observed by given by Woodward (1980). The other h values were treatment at pH 12.0; results not shown). Con- taken from the literature or calculated from the sequently, the concentrations used in the calculation published atomic co-ordinates (see Table 2). of the kinetic parameters for the interactions between penems and both fl-lactamases and D- alanyl-D-alanine-cleaving serine peptidases were Results 50% of the total concentrations. For comparison purposes (see the Discussion Non-planarity ofthe f3-lactam nitrogen and suscepti- section), benzylpenicillin and 6-aminopenicillanate bility of the f-lactam amide bond to nucleophilic were included in Tables 3 and 4. All the fl-lactam attack by OH- atpH 12.0 and 370C compounds considered were inactivators of the R61 Table 2 shows that depending on the families to and R39 peptidases except the three penems which which they belong, the fl-lactam compounds have were regarded as substrates of the same peptidases varying h values (h = distance between the fl-lactam (Table 5). Nevertheless, the data of Tables 3, 4 and 5 nitrogen and the plane formed by its three sub- can be compared with each other on the basis that: stituents). Table 2 also gives quantitative estimates (i) for the peptidases, kcat is equivalent to k+3 (see of the susceptibility of the fl-lactam carbonyl carbon the Materials and methods section and footnote to to nucleophilic attack by OH- at pH 12.0 and 370C. Table 5); and (ii) if the formation of complex El is a When the two procedures used yielded M-1* s-' (OH-) rapid-equilibrium process, then K, k+2 and k+3 are values differing from each other by a factor larger related to Km and kcat by Km = Kk+ 3/(k + k3) than 1.5, the individual values are reported. and kcat. = k+2k+3/(k+2+k+3) so that kcat./Km and 1982 fl-Lactamases and D-alanyl-D-alanine-cleaving serine peptidases 227

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Table 3. Interaction offi-lactam compounds with the R39 and G #-lactamases (at 30°C andpH7.0) R39 fl-lactamase G ,-lactamase

VM.Vmax. Vmax. Km (umol. min-I* kcat. kcat./Km Km (umol - min-I - kcat. kcat./Km f)-Lactam compounds (mM) mg of protein-') (s-') (M-'.S- (mM) mg of protein-') (S-') (M-' . s-) 2-Cephem (1) 0.3 30 7.5 25000 2.0 40 20 10000 3-Cephem (2) 0.4 12 3 7500 1.3 17 8.5 6500 Benzylpenicillin (13)(a) 0.065 256 64 980000 0.74 920 460 620000 6-Aminopenicillanate (12)(a) 0.07 200 50 700000 0.6 700 350 600000 Penicillanate (11) 0.11 56 14 127000 2.5 125 62 25000 Unsubstituted penem (22) 0.3 26 6.5 20000 0.6 670 335 1000000 2-Methylpenem (23) 0.25 66 16 64000 0.25 660 330 2500000 2-Phenylpenem (24) 0.05 30 7.5 150000 0.36 910 455 2500000 (a) From Kelly et al. ( 198 la).

Table 4. Interaction of /3-lactam compounds with the R61 and R39 D-alanyl-D-alanine-cleaving serine peptidases [at 37° C unless otherwise indicated and atpH7 (R61 peptidase) or 8 (R39peptidase)] R61 peptidase R39 peptidase Half-life of Half-life of k+2/K k+3 complex EI* k+2/K k+3 complex EI* ,B-Lactam compounds (M'.s-') (s-') (min) (w'.s-') (s-') (min) 2-Cephem (1) 68 3 x 10-3 4 52 <2x 10-s >575 3-Cephem (2) 77 SxlO-6 2270 4850 < xI 1O-7 >100000 Benzylpenicillin (13)(a) 14000 (25IC) 1.4 x 10-4 80 300000 2.8 x 10-6 4100 6-Aminopenicillanate (12)(b) 0.25 <6 x 10-s >2000 1200 5.6x10-3 2 Penicillanate (11) 0.06(c) 6 x 10-4 20 14 6x 10-4 20 (a) From Frere et al. (1973, 1974). (b) From Kelly et al. (198 lb). (") K = 36 mM; k+2 = 2.2 x 10-Is-' (see the Materials and methods section).

Table 5. Interaction of f-lactam compounds with the R61 and R39 D-alanyl-D-alanine-cleaving serine peptidases [at 370C andpH7 (R61 peptidase) or 8 (R39peptidase)] R61 peptidase R39 peptidase

Vmax. Vmax. Km (CMiol*min-'. kcat.(c) kcat./Km Km (,umol *min-l * k,,(c)t k+3(d) kcat./Km fJ-Lactam compounds (mM) mgaf protein-') (s-1) (M-l-s1) (mM) mg of protein-') (s-') (s-') (M- .S-I) Unsubstituted 0.08(a) 0.072 0.05 670 <0.05(a) 0.008 0.007 0.008 1750 penem (22) 0.07(b) 0.004(b) 2-Methylpenem (23) 0.125 0.015 0.01 800 <0.05(a) 0.009 0.008 0.008 5400 000050(b)1 2-Phenylpenem (24) 0.14 0.031 0.02 1400 <0.03(a) 0.011 0.010 0.010 10000 0.00I (b) (a) As obtained from Lineweaver-Burk plot (direct procedure). (b) As obtained from substrate-competition experiments with diacetyl-L-Lys-D-Ala-D-Ala. (c) As obtained from direct measurements. (d) As obtained from measurements of enzyme reactivation (indirect procedure). These values indicated that the EI* complexes have half-lives ranging between 70 and I00s. On the basis that kcat. = k,3 also applies to the interactions with the R61 peptidase, the EI* complexes formed with this latter enzyme have half-lives ranging between 14 and 70s. 1982 ,B-Lactamases and D-alanyl-D-alanine-cleaving serine peptidases 231 k+2/K are also equivalent. Also, on the basis that 0.04 (R39 peptidase) and those of 6-aminopeni- kcat. = k+3 and Km = kcat./(k+2/K), the Km values cillanate were <0.24 (R6 1 peptidase) and 0.05 (mM) of penicillinate were 10 (R61 peptidase) and (R39 peptidase). Finally, the Vmax. values (,umol * min- * mg of protein-') of penicillanate were 1X 10-3 (R61 peptidase) and 7x 10-4 (R39 pep- tidase), and those of 6-aminopenicillanate were <1 X 10-4 (R61 peptidase) and 9 x 10-3 (R39 peptidase). Comparisons between the second-order rate con- stants for (i) the hydrolysis of the f/-lactam amide bond by OH- and (ii) the opening of the same f/-lactam amide bond by the f/-lactamases and D-alanyl-D-alanine cleaving serine peptidases With the D-alanyl-D-alanine-cleaving serine peptidases, formation of the intermediate EI* involves the opening of the fl-lactam bond (and enzyme acylation) (Frere et al., 1976; Duez et al., 198 lb). Assuming that the fl-lactamases under study operate by the same or a similar mechanism, the second-order rate constants for hydrolysis of the fl-actam amide bond by OH- and the second-order rate constants (k+2/K or kcat /Km) for the opening of Wavelength (nm) the same fl-actam amide bond by both peptidases Fig. 1. Action of the R39 fi-lactamase on the racemic and fl-lactamases are comparable data (Table 6). 2-methylpenem The selected fl-lactam compounds (which include the Spectra were recorded at 30°C using a scanning 2- and 3-, penicillanate and three penems rate of 20nm/min (6 min for the whole spectrum) on under study) form five overlapping groups. Group a Beckman D-U 8 recording spectrophotometer. A, reveals the effects of a shift -.3- Spectrum of the intact 2-methylpenem (0.2mm, in a (a) 2-cephem total volume of 300,ul of 50mM-sodium phosphate, cephem -.penam (the three compounds having the pH7.0). The enzyme (lug in 5,ul) was then added, same aminoacyl substituent); group (b), the shift and spectra B, C, D and E were recorded succes- aminoacyl substitution -. amino substitution -+ no sively without interruption. No modification of substitution on C(6) of the penam nucleus; and group spectrum E was observed after several hours at (c), the shift 6-unsubstituted penam --various 6- 300C. unsubstituted penems. Groups (d) and (e) show the

Table 6. Second-order rate constants for the opening of the fi-lactam amide bond by OH-, f-lactamases and D-alanyl-D- alanine peptidases For explanation of the underlined values, see the Discussion section. R39 peptidase R61 peptidase at 37°C at 370C OH- R39 JJ-lactamase G fi-lactamase (M l *S l) (M- . S-') at 37°C at 300C at 300C (unless other- (unless other- f-Lactam compounds (M-l . S-1) (M-1.s-') (M-I-S-1) wise stated) wise stated) I 2-Cephem (1) 0.06-0.13 25000 10000 52 68 (a) 3-Cephem (2) 0.09-0.18 7500 6500 4850 77 Benzylpenicillin (13) 0.37 ± 0.10 980000 620000 300000 14000 (b) 6-Aminopenicillanate (12) 0.15 700000 600000 1200 0.25 Penicillanate (11) 0.01 127000 25000 14 0.06 (c) Unsubstituted penem (22) 1.0 20000 1000000 1750 670 2-Methylpenem (23) 0.5-1.25 64000 2500000 5400 800 + 2-Phenylpenem (24) 0.8-0.4 150000 2500000 10000 1400 (d) Cephalexin (3) 0.07 12300 1200 3000 (10°C) 4 Cephaloglycine (5) 0.70-1.1 50000 9200 74000 (200C) 22 (e)t Cephalothin (7) 0.42 + 0.12 300000 8800 >70000 3000 Nitrocefin (9) 3.0 2800000 930000 3600000 (10°C) 460 (10°C) * References for results for ,B-lactam compounds of groups (d) and (e): Frere et al. (1980); Duez et al. (198 la, 1982). Vol. 203 232 J.-M. Frere and others effects of various electron-withdrawing substituents tidases (and therefore the more readily this energy on C3) of the dihydrothiazine molecule in com- barrier is overcome). In this respect, examination of parable A3-cephalosporins. the data of Table 6 leads to the following ob- servations. (i) The accelerating effect due to enzyme action on a given JJ-lactam compound can be Discussion expressed by the ratio between the second-order rate The data of Table 2 strongly suggest that the constant for the opening of the fl-lactam amide bond degree of non-planarity of the fl-lactam nitrogen is by the enzyme under consideration and the second- mainly imposed by the nature of the ring that is order rate constant for hydrolysis by OH-, i.e. the fused to the fJ-lactam ring (and is relatively little ratio M-1 s-'(enzyme)/M-l S(OH-)1 Depending on influenced by the substituents that occur on both both the fl-lactam compound and the enzyme, this positions of the ring system). Pairs or groups of ratio varies from less than 2 (6-aminopenicillanate analogous or nearly analogous compounds can be and the R61 peptidase) to 1 x 106 or more. (ii) That selected, for which an increased pyramidal charac- a higher intrinsic reactivity of the 16-lactam com- ter of the Ilac*am nitrogen (as expressed by the h pound results in a faster formation of the inter- value) is accompanied by an increased lability of mediate EI* with a given f,-lactamase or D-alanyl- the ,J-lactam amide bond (as expressed by the D-alanine-cleaving serine peptidase depends on M-1 s-1(OH-) value). Penicillanate (compound 11), which f-lactam compounds are used for the com- for example, is 50-100 times less labile than the parison. Thus, with several groups of ,-lactam corresponding unsubstituted penem (compound 23). compounds and enzymes, the observed variations in It is dangerous, however, to generalize the rule. The the M-1. S-1(OH-) values, on the one hand, and the 2-cephem (compound 1) is only slightly more stable observed variations in the M-}- s- (enzyme) values, on than its 3-cephem analogue (compound 2). Mecil- the other, are roughly commensurate (at least within linam (compound 21) is 10 times more stable than one order of magnitude). This applies to the ,-lactam all the 'classical' (which, however, exhibit a compounds of groups (b), (d) and (e) and the R39 very similar h value). Doubt has already been cast J-lactamase, those of groups (b), (c) and (d) and the on the widely held view of the over-riding import- G fl-lactamase, those of groups (c), (d) and (e) and ance of the non-planarity of the 16-lactam nitrogen the R39 peptidase and those of group (d) and the (Gensmantel et al., 1981). Moreover, one should R6 1 peptidase (the relevant M-1 s- values are note (Pfaendler et al., 1981) that a carba-l-penem underlined in Table 6). In some cases, however, the (h = 0.54) is more stable than the analogous observed variations in the M-l *s-I(OH-) and the carba-2-penem (h = 0.50) and penem (h = 0.42- corresponding M-1 s-(enzyme) values are largely dis- 0.44). In the 3-cephem series, the electron-with- proportionate (compare the 2- and 3-cephems with drawing capacity of the substituent on C(3 seems to benzylpenicillin, whatever enzyme is considered). supersede any other structural feature [cf. ceph- Cases also exist where, contrary to the prediction, alexin and cephaloglycine (compounds 3 and 5), on increased M-1 * s-'(OH-) values are paralleled by the one hand, and cephalothin, cephaloridine and decreased M-1 s-(enzyme) values (compare the un- nitrocefin (compounds 7-9), on the other]. Remark- substituted and 2-methylpenems with penicillanic ably, also, cephaloridine is as reactive as the penems acid in their reaction with the R39 fl-lactamase, or (compounds 22-24), and nitrocefin, whose relevant nitrocefin with cephalothin in their interaction with geometry is very probably closely similar to that of the R61 peptidase). This strongly suggests that the the other 3-cephems, appears to be even more goodness of fit of the fJ-lactam molecule to the reactive than the thienamycin. The enzyme cavity, rather than its intrinsic reactivity, is chemical reactivity, in the ground-state conforma- the primary parameter that governs the enzyme tion, of the fl-lactam compounds thus appears to be catalytic efficiency. Other observations (Frere et al., the result of a complex interplay between the 1980; Ghuysen, 1980; Kelly et al., 1981a,b; Duez fused-ring system and its side chains. Within the et al., 1981a,b) lead to the same conclusions. limits of the present investigation, these combined Although the penams 13-19 (Table 2) have very effects result, at the most, in a 300-fold variation in similar M-1 5- (OH-) values, their M-I - s-'(enzyme) the second-order rate constants for hydrolysis of the values widely vary: from 15 () to 14000 fJ-lactam amide bond by OH- at 37°C, from (benzylpenicillin) with the R61 peptidase; from 1100 0.01 M-1 * s-' (penicillanate) to 3.0M-1 * s-' (nitro- (methicillin) to 300000 (benzylpenicillin) with the cefin). R39 peptidase; from 9200 (methicillin) to 635000 It is reasonable to propose that the higher the (benzylpenicillin) with the G /J-lactamase; and from intrinsic reactivity of the fl-lactam compounds, the 46 000 () to 980000 (benzylpenicillin) with lower the barrier energy to formation of the the R39 fJ-lactamase. Conversely, N-formimido- intermediate EI* during interaction with the ,B- ylthienamycin, which has a considerably higher lactamases and the D-alanyl-D-alanine-cleaving pep- intrinsic reactivity than benzylpenicillin but has a 1982 ,B-Lactamases and D-alanyl-D-alanine-cleaving serine peptidases 233

very distinct geometry, is characterized by relatively Blanpain, P., Laurent, G. & Durant, F. (1977a) Bull. Soc. low M-'-*5(enzyme) values: 1000 for the R61 pep- Chim. Belg. 86, 767-774 tidase, 10000 for the R39 peptidase, 3000 for the G Blanpain, P., Melebeck, M. & Durant, F. (1977b) Acta ,-lactamase and 2000 for the R39 /B-lactamase. Crystallogr. B33, 580-582 Boyd, D. B. & Lunn, W. M. W. (1979) J. Med. Chem. 22, From all these observations, it seems very likely that 778-784 the structures of the side-chains on the bicyclic Brocklehurst, K. (1979) Biochem. J. 181, 775-778 fused-ring systems are responsible for the relative de Koning, J. J., Kooreman, H. J., Tan, H. S. & Verweij, positioning of the ,J-lactam carbonyl group and the J. (1975)J. Org. Chem. 40, 1346-1347 active site serine hydroxy group in the case of the Dexter, D. D. & Van der Veen, J. M. (1978) J. Chem. D-alanyl-D-alanine-cleaving serine peptidases. Some Soc. Perkin Trans. 1, 185-190 side-chains can induce a catalytically active geo- Domiano, P., Nardelli, M., Balsamo, A., Macchia, B., metry, others cannot. The same mechanism prob- Macchia, F. & Meinardi, G. (1978) J. Chem. Soc. ably occurs with the ,B-lactamases studied (pre- Perkin Trans. 2, 1082-1087 liminary work strongly suggests that these f4lac- Domiano, P., Nardelli, M., Balsamo, A., Macchia, B. & Macchia, F. (1979) Acta Crystallogr. B35, 1363- tamases are also serine enzymes). 1372 A last comment deserves attention. Recent Duez, C., Frere, J. M., Klein, D., Noel, M., Ghuysen, developments have shown that the concept that J. M., Delcambe, L. & Dierickx, L, (1981a)1Biochem. 6-lactam compounds are substrates of the ,B-lac- J. 193, 75-82 tamases and inactivators of the D-alanyl-D-ala- Duez, C., Joris, B., Frere, J. M., Ghuysen, J. M. & Van nine-cleaving peptidases is an oversimplification. Beeumen, J. (198 lb) Biochem. J. 193, 83-86 Structural features in the fl-lactam molecules can Duez, C., Frere, J. M., Ghuysen, J. M., Van Beeumen, J., deeply affect the half-lives of the intermediates EI* Delcambe, L. & Dierickx (1982) Biochim. Biophys. formed with both types of enzymes. N-Formimido- Acta 700, 24-32 ylthienamycin is an inactivator of the R39 f,- Fisher, J. E., Charnas, R. L. & Knowles, J. R. (1978) lactamase (Kelly et al., 1981a), whereas, as shown Biochemistry 17, 2180-2184 Frere, J. M., Ghuysen, J. M., Perkins, H. R. & Nieto, M. in the present paper, the three penems studied (1973) Biochem. J. 135, 463-468 formed very short-lived intermediates (half-life from Frere, J. M., Moreno, R., Ghuysen, J. M., Perkins, H. R., 14 to 100s) with both R61 and R39 peptidases. Dierickx, L. & Delcambe, L. (1974) Biochem. J. 143, Also, with the fl-lactamases, some f,-lactam com- 233-240 pounds are known that behave both a substrates and Frere, J. M., Ghuysen, J. M. & Iwatsubo, M. (1975a) as inactivators; the reaction pathway appears to be Eur. J. Biochem. 57, 343-351 branched and the acyl-enzyme intermediate can Frere, J. M., Ghuysen, J. M. & Perkins, H. R. (1975b) undergo either simple hydrolysis or a rearrange- Eur. J. Biochem. 57, 353-359 ment that produces an inactivated enzyme (Fisher Frere, J. M., Duez, C., Ghuysen, J. M. & Vandekerkhove, J. (1976) FEBS Lett. 70, 257-260 et al., 1978; Kelly et al., 1981a). Frere, J. M., Duez, C., Dusart, J., Coyette, J., Leyh- Bouille, M., Ghuysen, J. M., Dideberg, 0. & Knox, J. This work was supported by an Action Concertee from (1980) in Enzyme Inhibitors as Drugs (Sandler, M., the Belgian Government (convention 79/84-Il), the ed.), pp. 183-207, MacMillan, London and Basingstoke Fonds de la Recherche Scientifique Medicale, Brussels, Fuad, N., Frere, J. M., Duez, C., Ghuysen, J. M. & Belgium (contract no. 3.4501.79) and the National Iwatsubo, M. (1976) Biochem. J. 155, 623-629 Institutes of Health, U.S.A. (contract no. 5 ROI Al Galdecki, Z. & Weifel, M. (1978) Acta Crystallogr. 13364-05). J. A. K. has been working at the University of A34, S90 Liege under a one-year research fellowship from the Gensmantel, N. P., McLellan, D., Morris, J. J., Page, M. United States Public Health Service, National Institute of I., Proctor, P. & Randahawa, G. S. (1981) in Recent Allergy and Infectious Diseases (grant 1 F32 Al Advances in the Chemistry of fi-Lactam Antibiotics: 05735-01). We are grateful to Dr. J. Lamotte-Brasseur 2nd International Symposium 1980 (Gregory, 0. I., (Service de Cristallographie approfondie, Universite de ed.), Special Edition no. 38, The Royal Society of Liege, Belgium), who kindly calculated some of the h Chemistry, London values of Table 2 from the atomic co-ordinates available Ghuysen, J. M. (1980) Top. Antibiot. Chem. 5, 9-117 in the literature. Gregory, 0. I. (ed.) (1981) Recent Advances in the Chemistry of /3-Lactam Antibiotics: 2nd International Symposium 1980, Special Edition no. 38, The Royal References Society of Chemistry, London Hamilton-Miller, J. M. T. & Smith, J. T. (eds.) (1979) Albers-Sch6nberg, G., Arison, B. H., Hensens, 0. D., f-Lactamases, Academic Press, New York Hirshfield, J., Hoogsteens, K., Kaczka, E., Rhodes, Hodgkin, D. C. & Maslen, E. N. (1961) Biochem. J. 79, R. E., Kahan, J. S., Kahan, F. M., Ratcliffe, R. W., 393-402 Walton, E., Ruswinkle, L. J., Morin, R. B. & Indelicato, J. M., Norvilas, T. T., Pfeiffer, R. R., Wheeler, Christensen, B. G. (1978) J. Am. Chem. Soc. 100, W. J. & Wilham, W. L. (1974) J. Med. Chem. 17, 6491-6499 523-527 Vol. 203 234 J.-M. Frere and others

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1982