Proc. Natl. Acad. Sci. USAVol. 93, pp. 1747-1752, March 1996Biochemistry

The catalytic mechanism of f3-lactamases: NMR titration of anactive-site lysine residue of the TEM-1 enzyme

(antibiotics/lysine pK/protein NMR/heteronuclear NMR)

CHRISTIAN DAMBLONt, XAVIER RAQUETt, LU-YUN LIANt, JOSETrE LAMOTTE-BRASSEURt, EVELINE FONZEt,PAULETFE CHARLIERt, GORDON C. K. ROBERTSt, AND JEAN-MARIE FREREt§tCentre d'Ingenierie des Proteines, Institut de Chimie, B6, Universite de Liege, B-4000 Sart-Tilman (Liege 1), Belgium; and tBiological Nuclear MagneticResonance Centre and Department of Biochemistry, University of Leicester, P.O. Box 138, University Road, Leicester, LE1 9HN, United Kingdom

Communicated by Kurt Wuthrich, Eidgenossische Technische Hochschule Honggerberg, Zurich, Switzerland, October 10, 1995

ABSTRACT ,B-Lactamases are widespread in the bacte-rial world, where they are responsible for resistance to pen-icillins, cephalosporins, and related compounds, currently themost widely used antibacterial agents. Detailed structural andmechanistic understanding of these enzymes can be expectedto guide the design of new antibacterial compounds resistantto their action. A number of high-resolution structures areavailable for class A 8-lactamases, whose catalytic mechanisminvolves the acylation of a serine residue at the active site. Theidentity of the general base which participates in the activa-tion of this serine residue during catalysis has been the subjectof controversy, both a lysine residue and a glutamic acidresidue having been proposed as candidates for this role. Wehave used the pH dependence of chemical modification ofe-amino groups by 2,4,6,-trinitrobenzenesulfonate and the pHdependence of the e-methylene 'H and '3C chemical shifts (inenzyme selectively labeled with [E-'3C]lysine) to estimate thepKa of the relevant lysine residue, lysine-73, of TEM-1 ,B-lac-tamase. Both methods show that the pKa of this residue is > 10,making it very unlikely that this residue could act as a protonacceptor in catalysis. An alternative mechanism in which thisrole is performed by glutamate-166 through an interveningwater molecule is described.

Penicillins, cephalosporins, and related 3-lactams representthe large majority of antibiotics in current use, thanks to theirgenerally high effectiveness as antibacterial agents, coupledwith their low incidence of adverse effects on eukaryoticorganisms. Resistance of pathogenic bacteria to f-lactams ismost commonly mediated by the action of the ,3-lactamases.The most common /-lactamases are the class A enzymes, suchas the clinically significant TEM-1 /3-lactamase (1, 2). Al-though a variety of compounds able to escape the hydrolyticaction of these enzymes-the so-called third-generationcephalosporins-have been introduced, numerous variants ofthe TEM ,B-lactamase exhibiting extended substrate profilesand capable of hydrolyzing the ",3-lactamase-resistant anti-biotics" have been isolated around the world (3). Recenthistory suggests that random mutations coupled with theselection pressure exerted by the sometimes excessive use ofantibiotics can, within a few years, result in the appearanceof enzymes which hydrolyze any newly introduced 3-lactam.The design of efficient antibacterial compounds cannot,therefore, rely on the substrate specificity of the enzymes,but must rely on an improved understanding of their com-mon catalytic mechanism.The catalytic pathway of class A 3-lactamases involves the

acylation of the active serine [Ser-70 in TEM-1 ,3-lactamase;ABL numbering (4)], followed by the hydrolysis of the esterbond formed in this first step (1). As in the serine proteases,

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

a general base in the active site is expected to participate incatalysis by accepting the proton from the crucial Ser-70residue during the formation of a transient tetrahedral inter-mediate. On the basis of crystallographic and mutagenesisstudies (5-7), two mechanisms have been proposed for f-lac-tamases in which two distinct residues are postulated to fulfillthis essential role. In the first mechanism (5, 6), the proton ofthe active-site serine (Ser-70) is transferred to the carboxylateof Glu-166 directly (5) or via a water molecule (6). In thesecond mechanism (7), the unprotonated side-chain aminogroup of Lys-73 acts as the general base. This implies anunusually low pKa for the s-amino group of this residue, whichis postulated to be due to a strongly positive environment in theactive site. We now describe chemical modification and NMRexperiments which indicate a "normal" pKa value of 10-10.5for the Lys-73 side chain, contrary to the predictions of thislatter mechanism. Thus, the only potential general base in theactive site is the carboxylate of Glu-166, and a mechanismincorporating this is outlined.

MATERIALS AND METHODS

Materials. Cefoxitin and imipenem were obtained fromMerck Sharp & Dohme; j3-iodopenicillanic acid was fromPfizer Central Research (Sandwich, Kent, U.K.); cephalothinwas from Lilly Research Laboratories (Indianapolis); andtemocillin was from Beecham Pharmaceuticals. These com-pounds were kindly donated by the respective companies.Acetic anhydride, 2,4,6-trinitrobenzenesulfonate (TNBS) andL-[1-'3C]lysine were purchased, respectively, from Aldrich,Sigma, and Eurisotop (Saint-Aubin, France).

Production and purification of the enzyme. The TEM-1,B-lactamase was produced by Escherichia coli RB791 (lacIqL8) (8) bearing the pTacII plasmid (9) and purified to homo-geneity by the method of Raquet et al. (10). About 30 mg ofenzyme was obtained per liter of culture, and the yield afterpurification was 80%.The labeled enzyme was prepared by growing the produc-

ing strain on a modified M9 minimal medium (11) at 37°C.At OD600 = 0.8, L-[s-13C]lysine was added to the medium ata final concentration of 100 mg/liter, and ,B-lactamasesynthesis was induced by adding isopropyl ,3-thiogalactosideat a final concentration of 0.3 mM. The cells were harvested4 hr later, and the protein was purified as described byRaquet et al. (10).Chemical Modifications. TNBS titration. TNBS (750 p,M)

was added to the enzyme (5 j,M) in 100 mM sodium boratebuffer, pH 9.9 at 40°C; for measurements of the pH depen-

Abbreviations: TNBS, 2,4,6-trinitrobenzenesulfonate; P3ip, j3-iodopenicil-lanate; HSQC, heteronuclear single quantum coherence; HMQC-NOESY, heteronuclear multiple quantum coherence-nuclear Over-hauser enhancement spectroscopy.§To whom reprint requests should be addressed.

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dence of the rate of modification, 100 mM borate or 100 mMphosphate was used in the pH ranges 8.0-10.75, and 10.9-11.3,respectively. The number of trinitrophenyl groups bound perenzyme molecule was deduced from the absorbance changes,using a As367 value of 10,550 M-1'cm-1 (12).

Acetylation with acetic anhydride. The partially acetylatedenzyme was prepared as follows. A 5 ,tM protein solution in50% saturated sodium acetate, pH 7.5, containing 1 mMimipenem or cefoxitin, was cooled in an ice bath. To achievecomplete acetylation of the protein, five 2-gl portions of aceticanhydride were added to the protein solution (10 ml) at 4°C,over a 1-hr period. The resulting solution was dialyzed over-night against 5 mM sodium phosphate, pH 7.00, to remove theprotecting substrate. The dialyzed acetylated enzyme was thentreated with 750 ,uM TNBS (final concentration).

j-Iodopenicillanate (p3ip) titration. P3ip specifically reacts withthe active-site serine, yielding an adduct which exhibits acharacteristic absorption maximum at 325 nm (13). A largeexcess of P3ip (1 mM) was added to the partially acetylatedenzyme (5 ,tM) in 50 mM sodium phosphate buffer, pH 11 at25°C. The resulting solution was dialyzed overnight against 50mM sodium phosphate buffer, pH 7, to remove the excess ofP3ip and its hydrolysis product, a dihydrothiazine. The A325/A280 ratio of the ,Bip-modified acetylated enzyme reflects theacylation of the active site serine residue by ,Bip. With theP3ip-modified unacetylated enzyme, this ratio was 0.47 (13).NMR Sample Preparation. The enzyme was dialyzed against

5 mM sodium phosphate (pH 5.8) and the solution wasfreeze-dried. The solid was then dissolved in 500 ,ul of 2H20and the pH* (uncorrected meter reading) was adjusted byusing NaO2H and 2HCl. Protein concentrations were deter-mined from the absorbance at 280 nm (13); the proteinconcentration of all samples was 1 mM.NMR Spectroscopy. All spectra were acquired on a Bruker

AMX600 spectrometer (600.13 MHz for 1H, 150.91 MHz for"3C) at a sample temperature of 293 K. Quadrature detectionin the F, dimension was achieved by using the TPPI method(14). Globally optimized alternating-phase rectangular pulses(GARP-1) (15) composite pulses were used during acquisitionfor 13C decoupling. Data were processed on an SGI Indigousing FELIX 2.3 (Biosym Technologies, San Diego).

'H spectra were collected with a spectral width of 7812.5 Hz,using 4096 complex points and a recycle time of 1.5 s. Theresidual water signal was suppressed by applying a low-powerpulse at the appropriate frequency during the recycle time.Before Fourier transformation, the data were zero-filled andmultiplied by a Gaussian window function. 'H chemical shiftswere referenced to 3-(trimethylsilyl)-propionic acid.

Heteronuclear single quantum coherence (HSQC) experi-ments (16) were performed at five different pH* values (5.8,9.0, 9.65, 10.4, and 11.4), with pulsed-field gradient watersuppression (75 G/cm for 2 ms). (Preliminary experiments hadindicated that no change occurred in the pH range 6.5-9.0.)Spectra were collected with a spectral width of 1805 Hz in the'H (F2) dimension, using 2048 complex points, and 754.5 Hz inthe "3C (F,) dimension. A total of 200 ti increments of 48transients each were collected. Before Fourier transformationthe data were zero-filled in both dimensions to give a final datamatrix of 2048 (1H) by 1024 (13C) real points. Gaussian windowfunctions were used in both dimensions. 13C chemical shiftswere referenced indirectly to tetramethylsilane (TMS), withexternal dioxane set at 67.4 ppm from TMS.The heteronuclear multiple quanta coherence-nuclear Over-

hauser enhancement spectroscopy (HMQC-NOESY) (17) wascarried out at pH* 9.0. The data were collected with a spectralwidth of 6024 Hz in the 'H (F2) dimension, using 2048 complexpoints, and 1509 Hz in the 13C (F,) dimension. The mixing timewas 120 ms. Presaturation of the 2H20 resonance was appliedduring the relaxation delay of 1.3 s. A total of 256 t, incrementsof 96 transients each were acquired. Before Fourier transforma-

tion the data were zero-filled in both dimensions to give a finaldata matrix of 2048 (1H) by 1024 (13C) real points. Gaussianwindow functions were used in both dimensions.

RESULTSThe TEM-1 f-lactamase contains a total of 11 lysine residues,of which two, Lys-73 and Lys-234, are located in the active site,while all the others are fully exposed to the solvent (18, 19).Reaction of the lysine s-amino groups and the terminala-amino group with TNBS yielded an absorbance change inagreement with the total number of amino groups in theenzyme (11 lysine residues and 1 terminal a-amino group; Fig.1 Upper). When the experiment was performed in the presenceof 1 mM imipenem or 1 mM cefoxitin, compounds which formrelatively stable acyl-enzymes (20, 21), the absorbance changeupon TNBS modification decreased by a factor of about 2/12,consistent with protection of the two active-site residues. In asecond experiment, the enzyme was first treated with aceticanhydride in the presence of 1 mM cefoxitin to acetylate theterminal a-amino group and all the lysine residues except thosein the active site. The enzyme recovered after dialysis exhibiteda kcat/Km value for cephaloridin decreased by 20-25% com-pared with the unmodified protein, but the pH dependence ofthis kinetic parameter was not significantly changed. Inac-tivation of this partially acetylated enzyme by P3ip gave thesame A325/A280 ratio as that observed with the unmodifiedprotein (13), at both pH 7 and pH 11.3, indicating that theactive-site serine residue of the modified protein could befully acylated by the specific active-site reagent and thus thatthe active site had not undergone any major perturbation.Reaction of the partially acetylated enzyme with TNBS ledto an absorbance change corresponding to the modificationof about two residues (Fig. 1 Upper), which must be the twoactive-site lysines.The rate of TNBS modification of lysine residues is pH

dependent according to Eq. 1, since reaction occurs with theunprotonated s-amino group:

kobs = kmax'Ka/([H+I + Ka), [1]

where kma, and Ka are, respectively, the pseudo-first-orderrate constant for modification of a fully deprotonated lysineside chain and the dissociation constant of the lysine s-am-monium group. Fig. 1 Lower shows that the pH dependenceof the modification rate of the two active-site lysine residuesin the partially acetylated enzyme is in good agreement withPKa values of about 10 for these two residues, Lys-73 andLys-234.The pKa values of the lysine residues were also estimated by

NMR spectroscopy, using enzyme labeled with [s-13C]lysine.Highly selective labeling of the lysine residues was achieved.'H-13C HSQC NMR spectra of the labeled protein wererecorded at a number of pH values between 5.8 and 11.4, andthe spectra at the two extremes ofpH are shown in Fig. 2 Upper;these spectra are of good quality for a protein of this size (30kDa). It is important to note that the enzyme is still in its nativeconformation at pH 11.4. There is no coalescence of reso-nances in the HSQC experiment at this pH, and structurallysensitive proton signals, such as high-field methyl resonances,are still present unaltered in the one-dimensional protonspectra (not shown).

Several sharp intense peaks were seen in the HSQC spectra,which appeared as clear triplets, particularly at low pH,implying magnetic equivalence of the C--methylene protons ofthe residues from which these peaks arise. It is clear from thepH dependence of the spectra that, at a given pH, some ofthese correspond to more than one lysine residue. In addition,four broader peaks were observed (labeled B1-B4 in Fig. 2).At all pH values, these latter peaks appeared in pairs (B1, B2

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Proc. Natl. Acad. Sci. USA 93 (1996) 1749

12 24 36Time, min

48 60

0

Peaks Bi and B2 were assigned to Lys-73 by means of an

HMQC-NOESY experiment, the results ofwhich are shown inFig. 3, in conjunction with the crystal structure. A cross sectionat the 13C frequency of the lysine C8-methylene resonances

(Bi, B2) reveals NOEs to a group of signals with 'H shifts of1.6-1.8 ppm, characteristic of the C6-methylene protons oflysine, and to a single resonance with a 'H chemical shift of 7.65ppm, characteristic of an aromatic residue. Examination of thestructure of TEM-1 P3-lactamase (Fig. 4) shows that theC6-methylene protons of Lys-73 are 2.35 A from the C6 protonof Phe-72, close enough to account for this observed NOE.None of the other 10 lysine residues is close enough to anaromatic ring to generate a similar NOE; the second-shortestsuch distance is that between Lys-34 and Phe-60, which is 5.3A. Even allowing for possible slight differences betweensolution and crystal states, this provides a clear-cut distinctionbetween Lys-73 and the other 10 lysines. We conclude that

12-

10~

.! 8-

£, 6-

4-

2-

6 7pH

FIG. 1. Modification of TEM-1 ,B-lactamase by TNBS. (Upper)Absorbance changes upon addition of TNBS to 5 ALM enzyme. 0,

Enzyme alone; v, enzyme protected by 1 mM cefoxitin; and A, enzymeacetylated in the presence of cefoxitin and dialyzed. The absorbancechanges after 60 min and deduced stoichiometries (Eo, total enzyme)were as follows: enzyme alone, AA = 0.65 and TNBS/Eo = 12.3;enzyme + cefoxitin, AA = 0.54 and TNBS/Eo = 10.2; partiallyacetylated enzyme, AA = 0.127 and TNBS/Eo = 2.4. (Lower) Rate ofTNBS modification of the partially acetylated enzyme as a function ofpH. The solid line represents the best fit to Eq. 1, yielding theparameters pKa = 10.0 and kmax = 12.7 M-1s-1.

and B3, B4) with the same 13C chemical shift but two different'H shifts. This suggests that (Bi, B2) and (B3, B4) eachcorrespond to a single lysine residue in which the Ce-methyleneprotons are nonequivalent. This nonequivalence, together withthe linewidth of these peaks, suggests that they correspond tothe two active-site lysine residues, which exhibit much lowertemperature factors in the crystal structure (19) and are thusprobably less mobile than the nine surface lysines. All theobserved peaks showed the same general behavior as a func-tion of pH, the upfield 'H and downfield 13C shifts withincreasing pH being characteristic of the deprotonation of the£-ammonium group. Although peaks B3 and B4 were difficultto identify at some pH values, the pH dependence of the 'Hand 13C chemical shifts of Bi and B2 could be clearly followed(Fig. 2 Lower) and was consistent with a pKa value greater than10 for the lysine side chain from which they arise.l

lIn Fig. 2 Lower, pH* indicates a direct meter reading in 2H20, notcorrected for the deuterium isotope effect of 0.4 unit on the glasselectrode. For nitrogen bases the isotope effect on the ionizationequilibrium is opposite to that on the glass electrode, so that thenumerical value of a pKa estimated from meter readings in 2H20 isclose, but probably not identical, to that in H20. Hence a slightdifference between the pKa values estimated from chemical modifi-cation in H20 and NMR in 2H20 is to be expected. In both cases,however, it is clear that the pKa of the residue of interest is >10.

3.0 2.81H chemical shift, ppm

E

Q

C.

Ea)

I

40.0

E

40.5 a

rFcn

C._

E

41.0 S0

-41.5

2.6

E

h40.8v-DL

-40.5 500

pH*

FIG. 2. (Upper) IH-13C HSQC spectra of [s-13C]lysine-labeledf3-lactamase at pH* 5.8 and pH* 11.4. (Lower) pH* dependence ofthe 1H and 13C chemical shifts of peaks Bi and B2 (arising fromLys-73). O and 0, 1H chemical shifts for Bi and B2, respectively; A,"3C chemical shifts for Bi and B2. The notation pH* indicates a

direct meter reading, not corrected for the deuterium isotope effectof 0.4 unit on the glass electrode. For nitrogen bases the isotopeeffect on the ionization equilibrium is opposite to that on the glasselectrode, so that a pKa estimated from meter readings in 2H20 isclose to that in H20.

1.12

0.94 DOl- ° 0

vv

gv0.58

14-

pH* 5.8

BX1'B2

B3 B4 pH 11.4

B3 B4

qni

4L

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1750 Biochemistry: Damblon et alP

8.0 7.0 6.0 5.0 4.0'H, ppm

j400'

40.02

_81

140.4

7.8 7.5 7.2

3.0 2.0

3.3 3.0 2.7'H, ppm

1.0

C

40.0

Eaa

40.2

j40.29

.140.4

1.8 1.5 1.2

FIG. 3. Observation of an NOEbetween the lysine s-CH2 protonsgiving rise to resonances Bi and B2(assigned to Lys-73) and an aro-matic proton, assigned to Phe-72,allowing the assignment of theseresonances. (a, b, and c) Contourplots of selected regions of thespectrum. (d) Cross section takenparallel to the 1H axis at the 13Cfrequency of the (Bi, B2) lysines-CH2 resonances at 40.29 ppm.

peaks Bi and B2 in the HSQC spectrum can be assigned toLys-73, and hence that this residue has a "normal" pKa of >10.

DISCUSSION

Residue Glu-166 was the first serious candidate proposed toplay the role of the general base which abstracts the protonfrom the hydroiyl group of the active serine residue of class AP3-lactamases in the acylation step (k2) and activates a watermolecule in the subsequent hydrolysis of the acyl-enzyme(deacylation, k3; Scheme I).

(x 2Lys 234

33

C) loop

k+, k2 k3E + Sz=± ES -ES*-E + P

k-,

Scheme I

Indeed, various site-specific mutations of this residue resultedin enzymes exhibiting significantly decreased acylation anddeacylation rates (5, 22, 23). Of particular interest was theGlu-166 ->Asp mutant, where the electrostatic situation in the

active site was barely modified but which exhibited k2 and k3values both decreased by similar factors of about 1000. How-

Lys 234

FIG. 4. Stereoview of the activesite of the TEM-1 3-lactamase,showing the side chains of the res-idues conserved in most class A/3-lactamases, together with Phe-72, which is found only in theT,EM-1 enzyme, and water mole-cule Wl. The distance between theclosest aromatic and the Lys-73s-CH2 protons is 2.35 A (brokenline).

4 p d40.29 ppm

I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~t I

h t.,.. .1 , ! "!

r-04

--dk ,- ;_

/

. r, . . .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Proc. Natl. Acad. Sci. USA 93 (1996)

CO) loo(p

...

., .. %,,.""I i... ... ., -,. ..., ,-,

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Proc. Natl. Acad. Sci. USA 93 (1996) 1751

Lys 73

Ser 130

Ser 70

N ct$ ~Ala 237

4 W

Glu 166

Lys 73

Ser 13

Lys 73

Ser70

Lys 73

Ser 1

Ser 70-C

NH

Ala 237

Lys 73 Lys 73

Ser 130

Ser70

NH

Ala 237 Ala 237

Glu 166

FIG. 5. Stereoviews of models of the Henri-Michaelis complex (Top), the tetrahedral intermediate (Middle), and the acyl enzyme (Bottom) forthe hydrolysis of benzylpenicillin by the TEM-1 ,B-lactamase. The solid circles represent oxygen atoms.

ever, the three-dimensional structure indicated that, in the freeenzyme, the distance between the serine hydroxyl and theglutamate carboxylate was somewhat too long to allow for aneasy transfer of the crucial proton (18, 24, 25). Moreover, othermutations of this residue in various class A enzymes (Glu-166-> Ala and Glu-166 -> Asn) resulted in an apparently specificimpairment of the deacylation reaction (26, 27).II This wasinterpreted by assuming an "asymmetric" mechanism, in whichthe role of general base was performed by Glu-166 in thedeacylation step but by another residue in the acylation step.Elucidation of the crystal structures of the Glu-166 -> Asnmutant of the TEM-1 enzyme and of the stable adduct formedwhen these crystals were soaked in a concentrated benzylpen-icillin solution led to the hypothesis that the deprotonated sidechain of Lys-73, a residue universally conserved in the Ser*-Xaa-Xaa-Lys sequence (where Ser* is the active-site serine)

IIt should be noted that the experimental conditions utilized to revealthe accumulation of the acyl-enzyme (7) were such that a 1000-folddecrease of the acylation rate in the Glu-166 -- Asn mutant would nothave been detected.

characteristic of active-site-serine penicillin-recognizing en-zymes, could act as a general base (7). This hypothesis implieda markedly decreased pKa value for the alkylammonium groupof this side chain compared with that of a "normal" lysineresidue. This unusual behavior was explained on the basis ofthe "highly positive" environment in the active site, resultingfrom the dipole of ca-helix 2 and the presence of the alkylam-monium group of another conserved lysine residue (Lys-234 atS A) in the immediate vicinity; this explanation, however,overlooked the negative charge of Glu-166 in the wild-typeenzyme even closer (3.4 A) to Lys-73. Very recently, Swarenet al. (28) have reported electrostatic calculations which leadto an estimate of 8.0 for the pKa of Lys-73 in the free enzyme,increasing markedly to 14.4 in the Michaelis complex. As theypoint out in the context of their estimates of the pKa of Ser-70,these calculations are very sensitive to the details of chargedistributions and local dielectric variations. It should be notedthat a pKa of -8 for a group which must be unprotonated formaximum activity has not been seen in the kcat/KM profiles ofany class A f3-lactamase. However, it is possible that themodified electrostatic situation in the active site due to the

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NHH

NH E1 NH

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Table 1. Rate constants for acylation of class A ,3-lactamases byvarious compounds

k2/K', mM-1 s-1

Streptomyces Bacillusalbus G licheniformis TEM-1

Substrate ,B-lactamase ,B-lactamase ,B-lactamase

Ticarcillin 125* 4700t 8600§Temocillin <0.1t 0.0023* <0.01Cephalothin 370* 2500t 650Cefoxitin <0.001t 0.02t 0.006w

kcat/Km = k2/K', where K' = (k 1 + k2)/k+1. Results are shown forcompounds containing a 6 (temocillin) or 7 (cefoxitin) a-methoxygroup and similar compounds (ticarcillin and cephalotin, respectively)devoid of such a substituent. *, t, t, §, and ¶ indicate data from refs.29, 30, 31, 23, and 21, respectively.

disappearance of the Glu-166 negative charge in the Glu-166-> Asn mutant might lead to a different mechanism, in whichthe Lys-73 side chain would exhibit a decreased pKa, allowingit to act as an alternate general base in catalysis by the mutantenzyme.The experimental data presented in the present paper

indicate that the pKa of the Lys-73 side chain is not significantlydecreased in the wild-type TEM-1 enzyme and that its sidechain bears a positive alkylammonium group in the pH rangewhere the enzyme exhibits optimal values of kcat/Km, a pa-rameter characteristic of the acylation step. It is thus unlikelythat this group is the general base in the acylation reaction.This leaves only one candidate for playing such a role: Glu-166.Although the distance between its carboxylate oxygens and thehydroxyl group of the active serine might appear to be too longto allow a direct proton transfer, a conserved water moleculebridging the two side chains offers a possible relay for thistransfer. Indeed, this water molecule, Wl, is found in an idealposition between the two side chains by both crystallographicand computer-simulation methods. Its importance is indicatedby the behavior of penicillins and cephalosporins containing amethoxy group on C6 (penicillins-e.g., temocillin) or C7(cephalosporins-e.g., cefoxitin). These compounds acylatethe active serine very slowly or not at all (depending upon theenzyme under study), while similar molecules lacking themethoxy group do so with rather high efficiencies (Table 1).Computer-aided modeling of the Henri-Michaelis complexesformed by these two types of ,B-lactams show that both caneasily be fitted into the enzyme active site and that the onlydifference is that the methoxy group of the former displacesthe water molecule, thus interrupting the proton-transferprocess (30). All these data support the model previouslyproposed (6), shown in Fig. 5, in which the proton, initiallyabstracted by Glu-166, is back-donated to the thiazolidineleaving group via an array of hydrogen bonds, in which the sidechains of Lys-73 and Ser-130 (another conserved residue)participate. Deacylation would then occur according to anearly symmetrical pathway, with the intervention of a second,exogenous, water molecule (6). An alternative, recently pro-posed, hypothesis is that the flexibility of the "omega-loop," onwhich Glu-166 is situated, might be sufficient to bring its sidechain near the Ser-70 hydroxyl (32).

This study was supported in part by the Belgian Government in theform of a P6le d'Attraction Interuniversitaire (PAI no. 19), an ActionConcertee with the Belgian Government (89-94/130), and the Fondsde la Recherche Scientifique Medicale (3.4531.92). The LeicesterBiological Nuclear Magnetic Resonance Centre is supported by theBiotechnology and Biological Sciences Research Council. The ex-

changes between Liege and Leicester were financially supported by theBritish Council, the Commissariat General aux Relations Internation-ales (Brussels), and the Fonds National de la Recherche Scientifique.The purchase of the SGI Indigo was financed by grants from theLoterie Nationale (Belgian Fonds National de la Recherche Scienti-fique no. 9.4585.92) and the University of Liege.

1. Waley, S. G. (1992) in The Chemistry of /3-Lactams, ed. Page,M. I. (Chapman and Hall, London), pp. 198-228.

2. Matagne, A. & Frere, J. M. (1995) Biochim. Biophys. Acta 1246,109-127.

3. Jacoby, G. A. & Medeiros, A. A. (1991) Antimicrob. AgentsChemother. 35, 1697-1704.

4. Ambler, R. P., Coulson, A. F., Frere, J. M., Ghuysen, J. M.,Jaurin, B., Joris, B., Levesque, R., Tiraby, G. & Waley, S. G.(1991) Biochem. J. 276, 269-272.

5. Gibson, R. M., Christensen, H. & Waley, S. G. (1990) Biochem.J. 272, 613-619.

6. Lamotte-Brasseur, J., Dive, G., Dideberg, O., Charlier, P., Frere,J. M. & Ghuysen, J. M. (1991) Biochem. J. 279, 213-221.

7. Strynadka, N. C. J., Adachi, H., Jensen, S. E., Johns, K., Sielecki,A., Betzel, C., Sutoh, K. & James, M. N. J. (1992) Nature(London) 359, 700-705.

8. Brent, R. & Ptashne, M. (1981) Proc. Natl. Acad. Sci. USA 78,4204-4208.

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