ISSN 1463-9076

Physical Chemistry Chemical Physics

www.rsc.org/pccp Volume 15 | Number 47 | 21 December 2013 | Pages 20381–20772

1463-9076(2013)15:47;1-F

PAPERChass et al.Penicillin’s catalytic mechanism revealed by inelastic neutrons and quantum chemical theory

This journal is c the Owner Societies 2013 Phys. Chem. Chem. Phys., 2013, 15, 20447--20455 20447

Cite this: Phys. Chem.Chem.Phys.,2013,15, 20447

Penicillin’s catalytic mechanism revealed by inelasticneutrons and quantum chemical theory†‡

Zoltan Mucsi,*a Gregory A. Chass,*b Peter Abranyi-Balogh,c Balazs Jojart,c

De-Cai Fang,d Annibal J. Ramirez-Cuesta,e Bela Viskolczc and Imre G. Csizmadiaa

Penicillin, travels through bodily fluids, targeting and acylatively inactivating enzymes responsible for

cell-wall synthesis in gram-positive bacteria. Somehow, it avoids metabolic degradation remaining inactive

en route. To resolve this ability to switch from a non-active, to a highly reactive form, we investigated the

dynamic structure–activity relationship of penicillin by inelastic neutron spectroscopy, reaction kinetics,

NMR and multi-scale theoretical modelling (QM/MM and post-HF ab initio). Results show that by a self-

activating physiological pH-dependent two-step proton-mediated process, penicillin changes geometry to

activate its irreversibly reactive acylation, facilitated by systemic intramolecular energy management and

cooperative vibrations. This dynamic mechanism is confirmed by the first ever reported characterisation of

an antibiotic by neutrons, achieved on the TOSCA instrument (ISIS facility, RAL, UK).

Introduction

The 20th century was a time filled with successful medicinaluse of – and improvement on – nature’s biomolecules,1 such asdinucleotide coenzymes (NAD and FAD),2 calicheamicin-(g1

l)1,3,4

duocarmycin,5–7 syringolin A,8 aflatoxin B1,9 penicillin.10

Although penicillin is recognised as one of the great discoveriesof the 2nd millennium,11,12 the appearance of resistant bacteriasignificantly reduced its utility and viability.12–15 Regardless,continued characterisation of its structure and mechanism aidsin the understanding and design of novel antibiotics withdesired effects and great precision.16

Penicillin hones-in on the bacterial transpeptidase enzyme,efficiently blocking cell wall synthesis. Since its discoveryby Fleming in 1928 and mass production by the British,10,17

it has remained a mystery as to how penicillin can escape thebody’s hydrolytic effects, while maintaining its ability to rapidly

acylate the transpeptidase’s serine side-chain oxygen.18,19

The ‘rare’ fused b-lactam-type structure of penicillin20 wascandidate to explain its reactivity, approximated to that of acidchlorides,21,22 but failed to account for stability under thehydrolytic conditions of body fluids. Modern works have focusedon and advanced understanding of bacterial resistance, whereinpenicillin hydrolysis is described;19–23 the mechanistic bases ofactivity having been proposed in the mid-20th century.20–22

However, penicillin’s change from a stable to a highly activecompound of directed-specificity has yet to be addressed – untilnow. This work opens the way for the 1st time to a quantitativeunderstanding of penicillin’s dynamic biochemical reactivity.

Herein, our hypothesis that penicillin is triggered by a self-activated, pH-dependent two-step protonation, is corroborated.As a result of the synergy between experiment and theory, weshow that penicillin functions as a system, much like moderncatalysts do.24,25 Specifically, an assembly of molecular compo-nents within a single molecule acting in unison to facilitate thedesired chemical transformation; pencillin effects an efficientacylation-inactivation of the transpeptidase enzyme. Structuralrequirements are equated to constructing the perfect mousetrap for catching targeted prey, while avoiding non-targets(Fig. 1).

Our combined experimental–theoretical approach opens theway to quantitatively characterising the systemic contributionsof the b-lactam amide bond and its role as a preferentialacylating agent, as well as that of the carboxylic acid functionality,present in all penicillin-type antibiotics (e.g. cephalosporine,26

thienamycin27,28). The functionality of the strained lactam-type

a Department of Chemistry, University of Toronto, Toronto, Ontario, M5S 3H6,

Canada. E-mail: zoltanmucsi@gmail.comb School of Biological and Chemical Sciences, Queen Mary University of London,

London, E1 4NS, UK. E-mail: g.chass@qmul.ac.ukc Department of Chemical Informatics University of Szeged, Szeged, H-6725,

Hungaryd College of Chemistry, Beijing Normal University, Beijing, 100875, Chinae ISIS Facility, STFC Rutherford Appleton Laboratory, Chilton, Didcot,

Oxon OX11 0QX, UK

† Dedicated to Sir Alexander Fleming on the 85th anniversary of his 1928discovery of penicillin.‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cp50868d

Received 27th February 2013,Accepted 28th May 2013

DOI: 10.1039/c3cp50868d

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ring system, as well as the difficult, yet requisite, and non-trivial,epimerisation (during penicillin biosynthesis) at the a-carbon(C7) of the L-valine residue,29–32 yet to be addressed, are alsocandidate for characterisation.

On the molecular scale, one of the simplest lethal weaponsis an acylating agent, easily reacting with amino acid side-chainfunctionalities (–OH, –NH2), including those essential to properenzyme function. The amide bond is one of the most appro-priate acylating functional groups, easily synthesised by living-organisms. However, to be lethal at the molecular level, itshould act immediately and irreversibly upon arrival. For thisreason, the typically non-active amide bond must somehow beactivated at the appropriate place and time. All of these implythat penicillin has an appropriate systemic assembly of func-tional groups to achieve this functionality. As a response toenvironmental change, from near-neutral body fluids to acidicactive site environs, the penicillinic system ‘‘shifts gear’’ from arelatively inactive to a highly reactive amide group. X-raycrystallography shows that the two fused rings are not inthe same plane, in addition to the amide bond being non-planar,20,21 resulting in reduced amide conjugation and thuspenicillin’s excellent acylating ability.

The question is therefore raised as to penicillin’s relativeactivity with respect to common acylating agents such as acylhalides or anhydrides. The reactivity, together with the strengthof the amide bond was estimated by the amidicity value,33–36

whereby, larger amidicity values generally correspond to morestable, less reactive amide linkages. Smaller amidicity valuescorrespond to lower stability and greater reactivity towardnucleophilic attack at the carbonyl carbon;33–36 concepts rele-vant to penicillin’s systemic molecular structure.

We therefore initiated a series of experimental and theore-tical characterisation of penicillin, including its cooperativelow-energy vibrations by inelastic neutron spectroscopy,towards furthering understanding of such systemic properties,to answer the following four questions:

QI: ‘‘How can an amide bond behave as a preferentialacylating agent and what is the carboxylic functionality’s role,present in all penicillin-type antibiotics?’’

QII: ‘‘Why such a strained lactam-type ring system isrequired for antibacterial action?’’

QIII: ‘‘Why the difficult epimerisation (during penicillinbiosynthesis) at the a-carbon (C7) of the L-valine residue29–32

is necessary for optimal activity?’’QIV: ‘‘How can organisms synthesise this highly active

compound, avoiding acylation of potent nucleophiles in bodyfluids, selectively activating and inactivating it as required?’’

MethodsNMR measurements

All 1H, 13C, 15N NMR and associated 2-dimensional (HSQC,HMBC, NOESY, COSY) NMR spectra were recorded on a BRUKERDRX-500 spectrometer in CDCl3. The molecular weight wasaccurately determined by UPLC-HRMS (Shimadzu) equipment.

Kinetic measurements

Hydrolysis of penicillin-G (referred to as penicillin fromhere on) (1), penicillin-G dimethylamide (4), 2-azetidinone (5)and dimethylacetamide (6), in a number of buffers (Table S1,ESI‡), and the rates of the reactions were measured by HPLC-UV-MS (AGILENT-1200). The integral ratio of the correspondingHPLC peaks and the benzoic acid was used to determine therate constant, using quasi 1st order approach (see Fig. S3, ESI‡).

Inelastic neutron spectroscopy

Inelastic neutron spectroscopy (INS) was conducted on theindirect geometry spectrometer TOSCA37,38 at the ISIS pulsedneutron and muon facility, Rutherford Appleton Laboratories,UK. Samples (B3 g) were loaded into aluminium sachets held inindium wire sealed thin walled aluminium cans, with datacollected over B6–15 hours, until a total of 1000 mA was collectedfor each sample. The INS spectra were generated from ab initiooutput (at the MP2(full) level) with the program ACLIMAX.39

Computational methods

Following rules within the context of systems chemistry,33–36

quantum chemical investigations were carried out with electronic

Fig. 1 A mouse trap (A), composed of bait (to entice appropriate prey), a sensor (to detect appropriate prey), a spring (energy reservoir to work) and a mortaltool (catching and killing the prey). The components are analogously reflected penicillin’s molecular structure (B), while the systemic aspects are reflected in amechanic model (C).

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structure computations, using the B3LYP40 and MP2(full)41

methods, employing the DGDZVP all-electron basis set42 for allatoms, using the Gaussian09 (G09) program package.43 All inputstructures were created using a standardised methodology.44

Electronic density analyses were completed using Bader’s estab-lished Atoms-In-Molecules (AIM) method.45,46

For docking using AutoDock 4.01,47 the 3D structure of theenzyme–ligand complex was downloaded from the PDB data-base, PDB ID: 1HVB.23 As only the reacted penicillin-inactivatesenzyme structure is available, and thus unsuitable for dockingcalculations, the covalent ligand-Ser62 bond was removed andthe beta-lactam ring re-cyclised, with requisite H-atoms addedvia Molecular Operating Environment 2006.08. The possibleH-bonding interactions were optimised via the PDB2PQR programpackage.48 The docking calculations resulted in one cluster,where the lowest and mean DGbind were �27.03 kJ mol�1, and�25.06 kJ mol�1, respectively (Fig. S5, ESI‡).

Amidicity (AM%) and resonance enthalpies [HRE(AM)]

The ‘‘amidicity scale’’, quantifying amide bond strength on alinear scale, based on the computed enthalpy of hydrogenation[DHH2

(AM); eqn (S6); Fig. S7A and B, Table S5, ESI‡] of the com-pound examined, comparing to reference compounds I and II. Themethodology is described in the ESI‡ and the literature.33–36

Results and discussion

To experimentally ascertain the impact of the cooperativeprotonation–deprotonation, amidicity and reactivity concept,the following experimental determinations were carried out:kinetic measurements, 15N & 13C NMR and inelastic (vibra-tional) neutron spectroscopy (INS).37,38 These were comple-mented by theoretical models of penicillin and its mechanismof acylation within the active site of the full bacterial trans-peptidase enzyme (1HVB, 347 amino acids) with 562 explicitwater molecules and one penicillin molecule (

P6833 atoms).23

Results emerging from this synergistic approach are detailed inthe following sub-sections.

Kinetic measurements & amidicity

Kinetic measurements were carried out at various pH values(Fig. 2) on penicillin [neutral (1) and anionic forms (2)],a synthetic non-deprotonatable penicillin derivate [penicillin-Gdimethylamide (4)] as well as on a four member lactone[2-azetidinone (5)]. These were subsequently compared to astandard amide compound [dimethylacetamide, DMA (6)]. Thehydrolysis of penicillin exhibits a near-identical hydrolytic rateunder acidic aqueous conditions as does the non-deprotonatablecompound (4). In basic conditions, the latter exhibits a signifi-cantly faster hydrolysis rate (200–1000 times faster) with respectto that of penicillin (1 and 2). The hydrolysis rate of the non-deprotonable form (5) is significantly slower in acidic media,and is unchanged in basic conditions with respect to 1 and 2,due to their similar amidicity values in basic media yetdissimilar values in acidic conditions. This (media-specific)phenomenon is schematically illustrated in Fig. 2 and labeled

as the catalytic effect of penicillin in acidic conditions and self-defensive (inactivation) effect in alkaline environs.

Towards explaining this dichotomy, the change in amidicity,thus reactivity, between penicillin’s anionic (1) and neutralforms (2) (Fig. 3) was explored. The inactive form (1), with itspronounced amidicity (51.8%) is more resistant to change(stable). Contrastingly, in the protonated semi-active form (2)the relative amidicity is reduced (17.2%), making it morereactive toward nucleophiles (–OH, –NH2), than (1).

The lowest energy conformer of neutral penicillin (2) is astable amide, thus sluggish acylating agent. Energetically, thenext most stable is an internally H-bound conformer (3), whichdespite exhibiting a higher energy content (2 - 3, 19.77 kJ mol�1

at the MP2(full)/DZVP level), has a much lower amidicity value(�36.5%), hence is more reactive. This very low amidicity value ison par with that of acetyl-pyridinium cation, a strong acylatingagent;33,34 reactive to most nucleophiles. The larger conforma-tional energy demand for this geometry significantly reduces thepopulation of this very active form of penicillin in the aqueousphase. It may however, become more energetically preferable inthe enzyme pocket (QIV).

The pronounced range of reactivity and amidicity of the threeforms (1, 2 and 3) were compared with some well-known amides.31

Fig. 2 Plot of the experimentally measured relative hydrolysis reaction rates fora strained amide bond (2-azetidinone, 5, black dashed line), penicillin-G dimethyl-amide (4, blue dashed line) and penicillin-G (1 or 2, red dashed line), as a functionof pH in aqueous media. The non-symmetric (about the pH = 7.0 line) curve ofpenicillin indicates faster hydrolysis in acidic, relative to basic, media. Thissignificant difference between the three curves is representative of the catalyticeffect for penicillin hydrolysis in acidic media. This is rationalised by the appear-ance of the far more active form (2), generated by protonation of the inactiveform (1). The superactive form (3) does not exist under these kinetic conditions.

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The activity of (1) is close to that of acylated imidazole (B59%) orbis-acyl compounds (B53%), well-known to be mild acylatingagents.

Proton-mediated activation & 15N/13C-NMR

In penicillin’s natural configuration, the COOH (2, 3) andCOO(�) (1) groups as well as the N-atom lone pair are on thesame face of the molecule (Fig. 3A). Deprotonation to form (1)the anion (COOH - COO(�)) is unfavorable, with a destabilising,repulsive interaction formed between the COO(�) and the lonepair of the N-atom (Fig. 3A); effectively pushing the N-atom into –and shortening – the amide bond, increasing its strength. This issupported experimentally by the larger dissociation constant(pKa = 2.7 � 0.1) observed for penicillin.

The acidity of (2) is large enough to be in its deprotonatedform (1) under physiological conditions (pH = 7.4), althoughless favourable than that of the corresponding a-amino acidssuch as valine and cysteine (pKa = 2.1–2.4), effectively decreasingthe acylating power of the lactam ring in (2).

Towards quantifying the dynamic protonation, the 15N and13C chemical shifts for the non- (1) and semi-active (2) forms of

penicillin were measured and correlated with the magnitude ofnet charges (d+, d�) resulting from the electron distribution inthe C–N bond of the beta-lactam ring (Fig. 3).

The 15N-NMR chemical shifts of for the N1 atom supports thehypothesis for reactivity difference between structures (1) and (2);15N-NMR shifts (in DMSO, 15N-HMBC, BRUKER 500DX, Fig. 3) of(2) (160.3 ppm) showed a 7.8 ppm upfield shift, with respect to (1)(168.1 ppm), indicating that the amide bond is stronger in (1),than in (2), in agreement with the pH-dependent activationmechanism proposed. Additionally, such a change of carboxylfunctionality leaves the chemical shifts of the other amide bondunchanged. These results were confirmed by theoretical charac-terisation of the electronic structures of (1), (2) and (3).

Electronic structure of penicillin

The electronic structures of penicillin salt (1), with Na+ counterion, as well as the semi-active (2) and active forms (3) of the acidwere determined using first principles (ab initio) quantumchemical theory [MP2(full) method, DGDZVP all-electron level].Results showed relative 15N chemical shifts of 8.2 and 10.3 ppmupfield for the semi-active (2) and active (3) forms, respectively,

Fig. 3 (A) The amidicity spectrum, showing the reactivity of the carbonyl group, of different penicillin forms 1, 2 and 3 with respect to that of alternate lactam-containing amides 4 and 5. Equilibrium between 1, 2 and 3 is also provided. The electron repulsion between the COO(�) at C7 and the lone pair at the N1 atom pushesthe N1 atom’s lone pair back into the amide bond, strengthening the amide bond in 1 (larger amidicity %) decreasing reactivity with respect to 2. In form 3, theinternal H-bond withdraws density from the amide bond, weakening it (extremely low amidicity %) leading to an extremely high reactivity toward nucleophiles.(B) The schematic overlay of the experimentally measured 15N spectrum (in DMSO) of structures 1 and 2, the significant shift (7.8 ppm) strongly supporting thetrend displayed in (A). Quantum chemical theory supports these results, with an 8.2 ppm shift predicted at the MP2(full)/DGDZVP level on protonation of penicillinsalt (1 - 2).

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with respect to the non-active form (1); in close agreement withthe experimental result of 7.8 ppm, providing confidence of theaccuracy of theoretical determinations.

Electronic density analyses using Bader’s established Atoms-In-Molecules (AIM) method45,46 (Fig. 4, upper portion) showedthe N–C(QO) bond in the b-lactam ring weakening by B9%(DHb) caused by a B5% reduction of electron density in thebond (Drb), on going from the non-active (Hb = �0.3953 a.u.,rb = 0.3044 e� bohr�3), through semi-active (Hb = �0.3826 a.u.,rb = 0.2983 e� bohr�3) to active forms (Hb = �0.3607 a.u., rb =0.2896 e� bohr�3). Hb and rb are the bond energy in atomicunits (a.u.) and the number of electrons (e�) per spatial volume,measured in cubic bohr (1 bohr = 0.529 Å), at the bond criticalpoint (BCP) between two atoms (N and C in this case), respectively.

This is qualitatively observable as a reduction of electronsharing between the two atoms (green-shaded ovals) withinthe isodensity contour plots in Fig. 4; these maps of electronicdensity distributions are known as 2-dimensional Laplacians.

It can be qualitatively appreciated in the plots that theelectronic density is reduced between the N and C atoms ofthe lactam-ring, and is due to the adjacent H� � �N bond formed(rb = 0.0285 e� bohr�3) upon activation of structure 3; in agreementwith previous observations. AIM analyses also revealed a weaklypolar CQO� � �H–C hydrogen-bonding interaction, strengtheningupon activation, between the methyl-group closest to theb-lactam ring in structures 1 (rb = 0.0082 e� bohr�3), 2 (rb =0.0084 e� bohr�3) and 3 (rb = 0.0100 e� bohr�3), indicating itsimportance to the intra-molecular electronic network – and

Fig. 4 (top) Electronic structure results emerging from Bader’s atoms-in-molecules (AIM) analyses of the wavefunctions generated from the MP2(full)/DGDZVPgeometry-optimised structures of the non- (1), semi- (2) and active-forms (3) of pencillin-G. The 2-dimensional Laplacian analyses (top) provide an ‘overhead’ type viewthrough contour mapping of the electronic density, showing the weakening of the beta-lactam N–C bond. (bottom) Comparative vibrational spectra over the 200–1500 cm�1 (B24.8–186.0 meV) range emerging from inelastic neutron spectroscopy (INS) and theoretical computations at the MP2(full)/DGDZVP level (MP2), for thesodium-salt (1) and acid forms of penicillin (2).

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thus cooperativity of vibrational modes within penicillin. Moredetailed information on Bader’s established methodology canbe found in the ESI‡ and the relevant references.

Intra-molecular cooperativity & dynamics

The vibrational spectra of penicillin sodium-salt (1) and acidsamples (2, 3) were characterised in the 20–1500 cm�1 range byINS on the direct geometry spectrometer TOSCA,37,38 and sub-sequently compared with the theory-predicted normal modes ofvibration, for structures 1, 2 and 3 (Fig. 4, lower portion).Excellent agreement was found throughout the 20–1500 cm�1

(E2.5–185 meV) range covered, particularly in the ‘fingerprint’region of the spectrum (B160–660 cm�1 E 20–80 meV),providing further strong support for the accuracy of the theore-tical results. The best agreement for the acid came from thesemi-active form (2), as expected due to the instability asso-ciated with the active form (3); supported by theory, with thefree-energy of 2 being 18.31 kJ mol�1 (189.77 meV) lower than 3.

With this confidence in hand, we investigated the molecularmotions of each of the three structures, with trends thereinstrongly supporting the pH-dependent catalysis mechanismproposed. The weakening of the b-lactam ring’s N–C(QO) bondshows the expected red-shifting (moving to lower energy) of thebond stretching motion upon activation (1 - 2 - 3), with anaverage 40–50 cm�1 reduction in each of the principal modescoupled to this vibration as listed in Table 1. The frequency ofthe stretch of the b-lactam ring’s carbonyl (CQO) moiety is alsoincluded, showing the expected blue-shifting (moving to higherenergy) upon activation (1834 - 1846 - 1857 cm�1), signalingan increase in bond strength and electronic density on the O-atom(corroborated by AIM analyses) and hence its acylating ability.

Vibrational analyses also revealed that each of the six N–C(QO) stretching modes was coupled to C–H bending andwagging modes in the two methyl groups on the 5-memberedring, suggesting that these two groups are connected to themechanism of activation. This is supported by establishedworks showing no activity for penicillin analogues with eitheror both methyl groups removed.49–52

Ring-strain & novel mechanism of action

Continuing with the mouse trap analogy, the penicillin systemshould internally store sufficient energy, not only to ‘catch’

(react with) the target, but also to ‘incapacitate’ it (reactirreversible); effected through ring-strain energy. Two Born–Haber cycles (Fig. S9, ESI‡) consisting of four isodesmic reac-tions, were characterised for structures (1) and (2) towardsquantifying their respective ring strains (in kJ mol�1). Thesecalculations revealed that the ring opening of the four-memberring, occurring during the irreversible ‘suicide’ action, relieves�75 kJ mol�1 of ring-strain for both the neutral and anionicforms; symbolised in the mechanical model (Fig. 1C). Despitetheir near-identical four-member ring strain energies, theamide group reactivities in (1) and (2) differ greatly from theother amides; details are presented in the ESI‡ (Fig. S9).This further highlights that systemic energy storage andmanagement are essential to (bio)reactivity.

When penicillin’s inactive form (1) docks in the active site ofthe target enzyme (Fig. 5), it forms a non-covalent inactiveenzyme-reactant Michaelis complex-1 (Fig. 5A), insufficientlyactive (amidicity = 51.8%) to acylate the enzyme’s serine sidechain. However, the spatial proximity of Histidine298–H+ caneasily protonate the carboxylate anion of penicillin passingthrough a proton transfer channel, Histidine298 - Tyrosine157 -

Tyrosine159 - HOH trans-protonation, mediated by Lysine65

(Fig. 5E), and yielding the non-covalent superactive Michaeliscomplex-3 (Fig. 5B).

This is confirmed by a QM/MM model [B3LYP/6-31G(d,p)//MM(UFF)], with the protonation being moderately endothermic(TSA - B B +70.0 kJ mol�1), activating penicillin through asignificant drop in amidicity [from 51.8% to (�36.5%)], pro-moted by the internal H-bond between the COOH at C7 and N1.Consequently, the b-lactam amide bond becomes as active(Fig. 3) as that of an extremely strong acylating agent (i.e.acetylpyridine, �30%). This structure (Fig. 5B) immediatelyreacts with the Serine62 side chain through a relatively low energytransition state (TSB - C) with a barrier of B92 kJ mol�1,yielding a tetrahedral intermediate.

This labile intermediate irreversibly reacts to the finalstructure (Fig. 5D) through a TS with a very low barrier(TSC - D B 22 kJ mol�1), yielding the inhibited, covalentlybound, inactivated enzyme of high stability (�86.2 kJ mol�1

more stable than the reactants). Strongly supporting these results,is the fact that the non-covalent enzyme Michaelis complex is notthe preferred enzyme structure, with +132.63 kJ mol�1 excessenergy – hence not formed.

Summary of results

Penicillin’s widely varied acylating ability is highly unexpectedfor such a small molecule. It is the key for an efficient self-protecting and self-activating acylating molecule, with thecarboxylic group acting as a ‘sensor’ (Fig. 1) (QI). This signifi-cant alteration of amidicity, triggered by anion protonationfollowed by conformational change, is essential for appropriatebiological activity. The carboxylic group functions as a switch,toggling the internal acylating potential of penicillin. Deproto-nation of (2) protects it from hydrolysis under physiologicalconditions, with target-initiated protonation and conforma-tional change (1 - 2 - 3) activating the acylation power.

Table 1 Six principal normal modes of vibration involving beta-lactam ring N–Cbond stretches (cm�1) for differing forms of penicillin, as determined by theoryand experiment (italics)

b-Lactam ring N–C bond stretch modes (cm�1)

CQOStructure 1st 2nd 3rd 4th 5th 6th

1 Theory 816 954 1132 1358 1365 1462 1834Experiment 819 953 1137 1350 1380 1469 1836

2 Theory 802 905 1124 1325 1331 1402 1846Experiment 793 908 1116 1304 1336 1388 1875

3 Theory 795 890 1090 1294 1315 1394 1857Experiment N/Aa

D1 - 3 �21 �64 �42 �64 �50 �68 +23

a Highly reactive intermediate.

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Fig. 5 A hybrid QM/MM model of penicillin in differing protonation states within the enzyme pocket, comprising an initial box (Ø, top left), the transpeptidaseenzyme (1HVB, 347 amino acids), 562 explicit water molecules, and one penicillin molecule (

P6833 atoms), computed with the oniom ‘layered’ model. The high

accuracy quantum mechanical (QM) layer (A–D), contains penicillin, 7 explicit water molecules and 6 side chain functional groups with one CH2 (backbone) for eachamino acid residues (

P105 atoms). The remainder of the enzyme and 555 solvating water molecules (

P6728 atoms) are treated by molecular mechanics. The initial

state after docking (A, top right) of the anionic penicillin form (1) into the enzyme pocket (inactive Michael complex-1) serves as an energetic reference point. In thisstate penicillin is inactive, due to its large amidicity value (17.2%). Anionic penicillin self-activates through potonation by Histidine298, achieved through a protontransfer channel (see E, bottom right) with a relatively low B70 kJ mol�1 barrier (TSA - B), generating a superactive Michael complex-3. Penicillin is superactive in thisform due to its extremely low amidicity value (–36.5%) and readily reacts with the Serine62 side chain (TSB - C B 92 kJ mol�1) yielding a high-energy tetrahedralintermediate (C, middle left). This structure is very rapidly hydrolysed through a relatively low barrier and irreversible transformation (TSC - D B 22 kJ mol�1) to formthe inhibited enzyme complex (D, bottom left). The specific proton transfer channel (E, bottom right) is comprised of the side chains of Histidine298, Tyrosine157,Tyrosine159, Lysine65 and 1 explicit water molecule; appropriate spatial orientation of penicillin’s COO(�) group is mediated by Arginine263. The overall computed free-energy profile of the catalytic transformations (in kJ mol�1) is also included (F, bottom).

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If penicillin was a static (non-switchable) molecule, we mightvisualize the following two extreme situations.

In the hypothetical case of an overly active penicillin analogue,the molecule may reactively decompose in the body prior toreaching and fulfilling its functional goal. Conversely, a moderatelyacylative penicillin would be stable enough to survive the aqueousmedia, but may not exhibit sufficient reactivity towards itsintended enzyme target. Such a structure–function dichotomyusing a media-dependent, tunable reagent simultaneously satisfiesthese opposing requirements in penicillin.

Appropriate spatial assembly of the molecular ‘instruments’ isalso requisite. During penicillin biosynthesis, the L-configurationof valine is changed to D- by the N-(5-amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthetase enzyme (Fig. S10, ESI‡). The funda-mental question arises, as to the purpose of this energy-intensiveepimerisation, requiring complex enzymatic machinery. Theanswer lies in the need to have one of the –COOH or –COO(�)

functional group C7 oxygen atoms near to the N-atom lone pairin structures (1) and (2), allowing for electron density to bepushed into the –CO–Nz bond, greatly reducing reactivity innucleophilic attack (Fig. 3). The resultant and energeticallyunfavourable COOH� � �N interaction makes the natural configu-ration [L,D,D (2)] 11 kJ mol�1 less stable (therefore more reactive)than the non-natural L,D,L configuration (Fig. S8, ESI‡).

On the bases of the accumulated mechanistic experience,the uncatalysed reaction is expected to have a higher energyrequirement for the rate determining step than that of the firstintermediate of the catalysed process (Fig. 5B). Our novelmechanism represents a step-change in adding the aspect ofpenicillin’s dynamic protonation state to the (basic) mecha-nism proposed by Woodward in 1966.22 The requisite proto-nation in the catalytically enhanced acylating power, is readilyachieved by one of the active site amino acid side chains. Thissequence-specific identifiable feature, coupled with the unusualstrained ring-structure, now appears (in hindsight), to have beenone of the clues to characterising the irreversible reaction.

Conclusions

Using a multi-disciplinary approach involving kinetics, NMR,inelastic neutrons and theory, we show herein that systemschemistry is at work in penicillin’s target-focused anti-bacterialmechanism. Specifically that its activity is linked to systemicproperties, including efficient energy management. Structuralrequirements may be compared to constructing the perfectmouse trap for catching targeted prey, while avoiding wastefullytrapping non-targets (Fig. 1).

Changes in internal amidicity33–34 reveal how penicillinprotects itself from spontaneous hydrolysis in the body, whilestoring its potential as a strong acylating agent, activated onlyupon approach to the target transpeptidase enzyme. Theinelastic (vibrational) neutron spectroscopic results, in close-agreement with first principles quantum chemical theory, showedprotonation of the carboxylic acid moiety associated with a weak-ening of the b-lactam ring N–C(QO) bond. Additionally, vibrationalcoupling of the lactam-ring CQO with the two geminal methyl

groups (next to sulphur), explain the loss of activity on removal ofeither of these groups in studies of penicillin analogues.

Future ‘retro’ structure–activity investigations, coupled withthe reproducible methodology presented herein, may beapplied to other related biomedical challenges, particularly inthe advancement of understanding and rational design of novelantibiotics and other pharmaceuticals. One may, for example,consider reversible bindings to be requisite in non-destructiveapplications while non-reversible ones would serve as mortaltools. It is foreseen that the advancements made within thiswork will help engineer a novel series of design rules to suchbiomedical ends.

Acknowledgements

We thank Eva Andre for technical support as well as Dr JohnTompkinson and Dr Stewart F. Parker (ISIS facility, RAL, UK)for experimental support. The authors thank GIOCOMMS(Toronto/Budapest/Beijing) for computational resources andsupporting international research exchanges. GAC acknowl-edges the support of the EPSRC, UK (EP/H030077/1 andEP/H030077/2) and the Royal Society (IE120096) and thanksProfessor Felix Fernandez-Alonso, Dr John Tompkinson,Dr Stewart F. Parker (ISIS, UK) and Dr Andrew Taylor (STFC, UK)for helpful discussions. DCF thanks the National NaturalScience Foundation of China (21073016), Scientific ResearchFoundation of Beijing Normal University (2009SC-1) for sup-port. BJ, BV and IGC acknowledge the support of the followingprojects: TAMOP-4.2.2.A-11/1/ KONV-2012-0047 ‘New Functionalmaterials, their biological and environmental answers’ andTAMOP-4.2.2.C-11/1/KONV-2012-0010 ‘Supercomputer – thenational virtual laboratory’.

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