# The Mechanisms of Catalysis by Metalloβ-Lactamases

Authors: Michael I. Page; Adriana Badarau
Journal: Bioinorganic Chemistry and Applications
 (2008)

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## Abstract

Class Bβ-lactamases or metallo-β-lactamases (MBLs) require zinc ions to catalyse the hydrolysis of β-lactam antibiotics such as penicillins, cephalosporins, carbapenems,  and cephamycins. There are no clinically useful inhibitors against MBLs which are responsible for the resistance of some bacteria to antibiotics. There are two metal-ion binding sites that have different zinc ligands but the exact roles of the metal-ion remain controversial,  and distinguishing between their relative importance is complex. The metal-ion can act as a Lewis acid by co-ordination to the β-lactam carbonyl oxygen to facilitate nucleophilic attack and stabilise the negative charge developed on this oxygen in the tetrahedral intermediate anion. The metal-ion also lowers the pKa of the directly co-ordinated water molecule so that the metal-bound hydroxide ion is a better nucleophile than water and is used to attack the β-lactam carbonyl carbon.  An intrinsic property of binuclear metallo hydrolytic enzymes that depend on a metal-bound water both as the attacking nucleophile and as a ligand for the second metal-ion is that this water molecule, which is consumed during hydrolysis of the substrate, has to be replaced to maintain the catalytic cycle. With MBL this is reflected in some unusual kinetic profiles.


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## Body

## 1. Introduction

Allβ-lactam antibiotics, such as penicillins (1) and cephalosporins (2), contain the four-membered β-lactam
ring which occurs relatively rarely in nature, therefore it is not surprising
that the biological activity of these compounds should be attributed to the expected
enhanced chemical reactivity of the β-lactam ring [1]. It was suggested early
that the antibiotic’s activity was due to the inherent strain of the
four-membered ring or to reduced amide-resonance.  The nonplanar butterfly shape of the
penicillin molecule (3) was
expected to reduce amide-resonance and thus increase the susceptibility of the
carbonyl group to nucleophilic attack, compared with normal, planar amides [1]. However, it was shown later that there is little evidence to confirm that
the reactivity of β-lactams in penicillins and cephalosporins is due to an
unusually strained or an amide-resonance inhibited β-lactam [2]. In fact, the
rate of alkaline hydrolysis of the simple β-lactam (4) is only 3-fold greater than that of (5).(1)(2)(3)(4)(5)Most
nucleophilic substitution reactions ofβ-lactams involve initial covalent bond
formation between the carbonyl carbon the of β-lactam and the attacking nucleophile followed
by C–N bond fission of the β-lactam (Scheme
1).Scheme 1This
type of reaction is a two-step process and involves the reversible formation of
a tetrahedral intermediate [1, 2]. Contrary to expectations, opening the
four-membered ring is not a facile process [3] and, indeed,  in many of these nucleophilic substitution
reactions, the rate limiting step is not the first addition step but a
subsequent one which may sometimes even be ring opening itself [1, 2, 4–8].Another
interesting difference between nucleophilic substitution in penicillins and
peptides/amides is the preferred direction of attack and the geometry of the
initially formed tetrahedral intermediate. It is usually assumed that
nucleophilic attack on the carbonyl carbon of a planar peptide will generate a
tetrahedral intermediate with the lone pair on nitrogenanti to the incoming nucleophile, (6), (7).  Conversely, nucleophilic attack on the β-lactams
of penicillins occurs from the least hindered α-face (exo)
so that the β-lactam nitrogen lone pair is syn to the incoming nucleophile in the tetrahedral intermediate (8) [9]. This has obvious consequences for the placement of catalytic
groups—particularly, if catalysis involves
coordination to metal-ions [10].(6)(7)(8)


## 2.β-Lactamases

It is more than sixty years sinceβ-lactams were first introduced
into clinical practice, but their therapeutic importance remains extremely high
and they account for around half of total antibiotic prescriptions
[11]. Given the rapid replication rates of bacteria against this selective
pressure, it is not surprising that mechanisms of resistance continue to evolve
and spread amongst pathogenic bacteria. Although there are several mechanisms
of β-lactam resistance including modification of the target (transpeptidases), reduction
in permeability, and efflux of the antibiotic, the major reason for resistance is
antibiotic inactivation by the action of a variety of β-lactamases that are
found in a large number of clinically important pathogens [12–14]. This number presently
exceeds 500 and is growing http://www.lahey.org/studies/.β-Lactamases
catalyse the hydrolysis of the β-lactam of penicillins (1) and cephalosporins (2)
to give the ring opened and bacterially inert β-amino acid (Scheme 2) [15].Scheme 2The main mechanistic division ofβ-lactamases is into serine enzymes and zinc
enzymes [15]. The former have an active
site serine residue and the catalytic mechanism involves the formation of an
acyl-enzyme intermediate. The metallo enzymes appear to involve coordination of
the substrate and intermediates to the active site metal-ion. On the basis of
their amino acid sequences, the serine β-lactamases are subdivided into three
classes: A, C, and D, whereas the class B β-lactamases consist of the zinc
enzymes [15].


## 3. Metalloβ-Lactamases

Class Bβ-lactamases or
metallo-β-lactamases (MBLs) require zinc-ions to catalyse the hydrolysis of
β-lactams and have no sequence or structural homology to the serine
β-lactamases. They exhibit a broad spectrum substrate profile catalysing the
hydrolysis of a wide range of β-lactam antibiotics including penicillins,
cephalosporins, carbapenems, cephamycins,
and even some mechanism-based inhibitors of class A β-lactamases
[16]. The first metallo-β-lactamase to be discovered was produced by an
innocuous strain of Bacillus cereus,
but in the last 20 years, MBL-mediated resistance has appeared in several
pathogenic strains and is being rapidly spread by horizontal transfer,
involving both plasmid and integron-borne genetic elements [17].  MBLs represent a huge potential clinical
threat to the β-lactam antibiotic therapy as presently there are no clinically
useful inhibitors for them.MBLs
can be divided in three subclasses: subclass B1, B2, and B3 based on their
aminoacid sequences, substrate profile, and metal-ion requirement [18, 19].
Subclass B1 is the largest and contains several well-studied  β-lactamases: BCII from Bacillus
cereus, CcrA from Bacteroides
fragilis, IMP-1, SPM-1 from Pseudomonas aeruginosa, and BlaB from Cryseobacterium meningosepticum. These
enzymes efficiently catalyse the hydrolysis of a wide range of substrates such
as penicillins, cephalosporins, and carbapenems. BCII catalyses the hydrolysis of penicillins at significantly higher rates than
cephalosporins and carbapenems, but CcrA does not show this preference, although
both enzymes exhibit lower Km values for cephalosporins [20].The most common representatives of
subclass B2 are CphA fromAeromonas
hydrophila and ImiS from Aeromonas
veronii, which preferentially hydrolyze carbapenems, for example, imipenem
and meropenem, but have poor activity against penicillins and cephalosporins
[20, 21]. Finally, subclass B3 contains
the only known tetrameric zinc β-lactamase,
the L1 enzyme from Stenotrophomonas
maltophilia, and the monomeric FEZ-1 from Legionella gormanii. Both enzymes hydrolyze a wide range of
β-lactam antibiotics [22, 23] with L1 having higher catalytic rate
constants (kcat) for penicillins compared with FEZ-1, which shows
higher kcat values for cephalosporins. Generally, the Km values are smaller for L1 than for FEZ-1. However, mutation of a methionine
residue in L1, which is important for the subunit interaction, gives a monomeric
enzyme with significantly higher Km values and, except for
nitrocefin, smaller kcat values compared with the wild-type tetramer
[24].


### 3.1. The Number of Zinc-Ions

A major problem with
understanding the mechanism of MBLs is the number of zinc-ions required for
catalysis, which has been addressed by studying MBLs under both equilibrium and
kinetic conditions. The crystal structures of several MBLs have been determined
by X-ray diffraction and they all  show a
similarαβ/βα sandwich fold, which was first seen with MBLs
[25], but since recognised in other enzymes, such as glyoxalase II, aryl
sulfatase, and cAMP phosphodiesterase, which have
now become members of the MBL fold superfamily [26]. The active site of MBLs is
situated at the bottom of a wide shallow groove between two β-sheets and has two potential zinc-ion binding sites at
the active site, often referred to as sites 1 and 2 [27–29]. The zinc ligands
in the two sites are not the same and are not fully conserved between the
different MBLs. Table 1 shows
the enzyme residues involved in zinc coordination in the two binding sites in
the subclasses B1, B2, and B3 [26].Table 1
The zinc
ligands in class Bβ-lactamases.
SubclassZn1 ligandsZn2 ligandsB1His116His118His196Asp120Cys221His263B2Asn116His118His196Asp120Cys221His263B3His/Gln116His118His196Asp120His121His263In the subclass B1, such as
theBacillus cereus enzyme BCII, the zinc in site 1 (the histidine site or His3 site) is tetracoordinated by the imidazole groups of three histidine
residues (116, 118, and 196) and a water molecule, Wat1.  In site 2 (or the Cys site) the metal is
pentacoordinated by His263, Asp120, Cys221, and one water molecule; the fifth
ligand at site 2 is carbonate or water, often referred to as the apical water,
or Wat2 [27, 28, 30], although this is missing in structures with
inhibitors bound [31]. The two metal-ions are relatively close to each other,
but the distance between them varies from 3.4 to 4.4 Å  in different structures
of the BCII and CcrA enzymes.  Several structures of the CcrA enzyme show a
bridging water ligand between the two metals, which is thought to exist as a
hydroxide-ion [32]. In a structure of BCII determined at pH 7.5 that contains two zinc-ions there is also a similar bridging water molecule, but in structures of this enzyme at lower pH this
solvent molecule is strongly associated to the zinc in site 1 [30].The two conserved zinc
binding sites in MBLs have different metal-ion affinities. For example, the BCII enzyme fromBacillus cereus has very different dissociation constants for the
two metal binding sites. The first crystal structure, obtained at low pH [25], indicated
only one zinc-ion bound to the histidine site, but equilibrium dialysis studies
showed two binding events, with a dissociation constant of 0.3 μM for the first
zinc-ion (Kmono) and 3 μM for the second zinc-ion (Kbi)
[33]. Later metal binding studies by fluorescence spectroscopy using a
chromophoric chelator reported a Kmono of 0.62 nM and a Kbi of 1.5 μM [34]. As the conditions of the experiments were similar for the two
studies, the large discrepancy in Kmono may be due to the fact that
different strains of BCII enzyme
from Bacillus cereus were used. The dissociation
constants Kmono and Kbi have been determined from the
steady state rates of hydrolysis of imipenem in the presence of EDTA as a metal-ion
buffer at different zinc concentrations and Kmono was found to
decrease significantly, from nM to pM, in presence of substrate, whereas Kbi decreased only by two fold [35]. This led to the suggestion that the monozinc
enzyme is responsible for the catalytic activity under physiological
conditions, where the concentration of the “free" Zn2+ is in the pM or
even fM region. For most substrates, the reported catalytic activity of the
monozinc BCII was about two fold
lower than that of the binuclear enzyme. Conversely, the other class
B1 enzyme, CcrA
from Bacteroides fragilis, binds both
zinc-ions very tightly [36].  Despite the
very close similarity with BCII,
CcrA has much higher affinity for the second zinc-ion, probably due to the fact
that in CcrA, a cysteine residue replaces the positively charged arginine 121
found in BCII.  However, replacing Arg 121 in BCII by a Cys shows no increase in the affinity for
the second zinc-ion [37].Early kinetic studies of CcrA
led to the proposal thatboth the
mono and the dinuclear forms of the enzyme were catalytically active, with
slightly different activities, at physiological pH [38]. However, later studies showed that only the
dinuclear species was active and that the previously observed “monozinc” CcrA
was a mixture of the dizinc and the apo (metal free) enzyme [39]. Class
B2 metallo β-lactamases are catalytically active with one bound zinc-ion and
the binding of the second zinc-ion inhibits the enzyme noncompetitively, with a
Ki of 50 μM [40]. The dissociation constant of the first zinc-ion was
found to be 7.0 and 1.2 pM in the absence and presence of substrate (imipenem),
respectively [35].  Subclass B3 MBLs, L1,
and FEZ-1 bind both metal-ions tightly, the dissociation constants are in the
nM region, and are fully catalytically active in binuclear form [23, 41].


### 3.2. The Catalytic Role of Zinc

Zinc is an essential trace element
and the second most abundant transition metal found in living sytems. Its role
in catalysis is related to its ability to participate in tight, but readily exchangeable
ligand binding and its exceptional flexibility of its coordination number and geometry
[42]. In addition, zinc shows no redox properties and this facilitates its
evolution in living systems without the risk of oxidative damage. Finally, its intermediate
hard-soft behaviour allows it to bind a variety of atoms, as seen, for example,
in the second binding site of class B1 MBLs which involve nitrogen, oxygen, and
sulphur as ligands. The Lewis acidity, flexible geometry, and coordination
number and the lack of redox properties make zinc an ideal metal cofactor for many
enzymes. The small energy difference between 4, 5 or 6 coordination geometries
and the rapid exchange of the kinetically labile zinc-bound water molecule are
important features in all zinc hydrolases including MBLs.MBLs
are a subclass of metallo-proteases and many mechanistic considerations are
applicable to both groups. There are many potential mechanistic roles for the
metal-ion in metallo-proteases and MBLs, although they may well vary from
enzyme to enzyme [42, 43]. It is commonly suggested that the metal-ion acts as
a Lewis acid by coordination to the peptide carbonyl oxygen giving a more
electron deficient carbonyl carbon which facilitates nucleophilic attack. The
metal-ion thus stabilises the negative charge developed on the carbonyl oxygen
of the tetrahedral intermediate anion (Scheme
3). Many metallo-proteases have a water molecule directly coordinated to
the metal-ion which may act as the nucleophile to attack the carbonyl
carbon.  Here, the role of the metal-ion
is to lower the pKa of the coordinated water so that the
concentration of metal-bound hydroxide ion is increased relative to bulk
solvent hydroxide-ion at neutral pH and, furthermore, although this coordinated
hydroxide-ion is different in nature than the simple solvated ion, it is a
better nucleophile than water (Scheme 4).
These two steps shown in Schemes 3 and 4 relate to
formation of the tetrahedral intermediate and although little attention is
normally given to the mechanism of the breakdown of the tetrahedral
intermediate, C–N bond fission is the most energetically difficult process in
peptide hydrolysis. This could be facilitated by direct coordination of the
departing amine nitrogen to the metal-ion (Scheme 5) which is, in
fact, the mechanism adopted for the simple zinc-ion-catalysed hydrolysis of
penicillin [10]. Alternatively, a metal-bound water could act as a general acid
catalyst protonating the amine nitrogen leaving group to facilitate C–N bond
fission (Scheme 6). Despite
intense mechanistic studies, the detailed roles of the metal-ion in
metallo-proteases and MBLs remain controversial and distinguishing between the
relative importance of the possible roles for zinc is complex.Scheme 3Scheme 4Scheme 5Scheme 6The effective positive charge on the zinc-ion depends on the number and nature of
its ligands and for those with an ionisable hydrogen, zinc lowers theirpKa and the ionised ligand obviously is better at neutralising the positive charge
density on the metal. The pKa of water is 15.7, but when bound to zinc
in aqueous solution it is lowered to 9.5, but is there evolutionary pressure to
lower the pKa of the zinc-bound water in an enzyme even further? This
could be achieved by replacing
say a negatively charged carboxylate ligand by a  neutral histidine. For example, in the
opposite direction, changing the histidine bound to zinc in carbonic anhydrase to
aspartate increases the pKa of the zinc-bound water from 6.8 to ≥ 9.6
[45]. Whether it is better to have a higher or lower pKa metal-bound
water for a faster reaction and more efficient catalysis depends on the role of
the zinc-ion. A low pKa implies a more electron deficient metal-ion
centre which would give a better Lewis acid to stabilise the negative charge
developed on the oxyanion of the tetrahedral intermediate. Conversely, a high
pKa for the metal-bound water implies a weaker Lewis acid and so the
zinc-ion will be less efficient at stabilising the tetrahedral intermediate. A
low pKa for the metal-bound water implies that the hydroxide-ion resulting
from ionisation is more “tightly bound” and stabilised which, although it
becomes the dominant species even at low pH, corresponds to a more weakly
nucleophilic hydroxide-ion. For example, if the pKa of the zinc-bound
water is about 5 then the nucleophilicity of the metal bound hydroxide-ion is
only similar to that of a carboxylate anion. If a major role of the metal-ion
is to provide a better nucleophile than water then the net effect depends on
the relative importance of concentration and the dependence of the rate upon
nucleophilicity. If the pKa is “too high”, metal-coordinated water
will be the dominant species over the desired pH range but deprotonation will
give a more nucleophilic metal-bound hydroxide. How reactivity changes with
changing pKa and pH will depend on the susceptibility of the rate of
reaction to the basicity of the nucleophile—the hydroxide-ion bound to the metal—as indicated by the Bronsted βnuc value.Sometimes it is suggested that part of the enzyme mechanism involves general base
catalysis to remove the proton from the zinc-bound water but this only becomes
necessary if thepKa is little changed from that of bulk water. A
problem with the commonly accepted mechanism for metallo-proteases of rate-limiting
deprotonation of zinc-bound water concerted with nucleophilic attack is the pKa of this water.  Coordination to zinc(II) generally
lowers the pKa of water to 5–9 depending on the number and type of
other ligands.  The concentration of
zinc-bound hydroxide ion is therefore quite high over the normal pH range
studied. There is little or no catalytic advantage in having a general base
to remove a proton in this pre-equilibrium step!  Even if the pKa of the
zinc(II)-bound water is about 9 then 10% of the species already exists in the
fully deprotonated form at pH 8 and 1% at pH 7.
Presumably, the deprotonated form is a much better nucleophile than the
species which is only partially deprotonated and there would be no catalytic
advantage of the general base-catalysed mechanism.In
aqueous solution, zinc is coordinated to six water molecules and thepKa of the zinc-bound water is 9.5. However, within a protein this pKa can be changed significantly because of the environment and directly bonded
ligands to the metal—for example, the zinc of B. cereusβ-lactamase is coordinated to three protein ligands in
the zinc1 site—His 116, His 118, and His 196 and a water molecule [30]. The
reduced coordination number of four in the zinc1 site of β-lactamase reduces the pKa to less
than 6. In principle, one or more of the imidazole residues could be ionised,
the pKa for proton loss from imidazole is similar to that of water (about
14). However, NMR evidence suggests that the imidazole of the bound histidines
are neutral [46, 47] and the most likely group to ionise is, therefore, the bound
water (Scheme 7).Scheme 7One
way to investigate the role of the zinc in catalysis is to modify thepKa of the zinc-bound water by changing the ligands or the metal-ion. If the
activity of the resulting enzyme increases as the pKa of the zinc-bound water ligand decreases, this would suggest that a high positive charge density
on the metal-ion facilitates catalysis and that the zinc coordination to the
carbonyl oxygen and stabilisation of the negative charge developed on this
oxygen following nucleophilic attack is important. A lower pKa of
zinc-bound water indicates a more electrophilic zinc which is better at
stabilising negative charge giving rise to a better catalyst. Stabilisation of
the intermediate anion must be more important
than the nucleophilicity of the zinc hydroxide.  Conversely, if activity increases with
increasing   pKa of the zinc-bound water, that is,
increasing basicity of the of the zinc-bound hydroxide ion, then this would
indicate a greater role for the metal-ion in controlling the nucleophilicity of
the hydroxide-ion compared with its role in stabilising the negative charge
development on the carbonyl oxygen.Thedual role of zinc in metallo-proteases, that is, acting as a Lewis
acid in polarising the carbonyl bond and as source of nucleophilic hydroxide,
requires formally the formation of an apparently strained four-membered ring (Scheme 8). However, this coordination occurs in the
bidentate complexes of zinc with carboxylate anions. Monodentate complexes
between zinc and a carboxylate anion exist predominantly as the syn stereoisomer due to the more basic syn lone pair and favourable opposition
of the C=O and O–Zn dipoles. The coordination adopted for the syn conformation is similar to a
bidentate one which is favoured by electrostatic interactions [48].Scheme 8One
final point to consider for the hydrolysis ofβ-lactams by metallo-β-lactamases is that if the zinc bound hydroxide-ion
is the nucleophile then it is consumed during turnover. Consequently, regeneration of the catalyst requires the
coordination of a new water molecule to the active site zinc. For example, if
product release occurs through displacement by water or is introduced in a
separate step following product release, deprotonation must occur to generate a catalytically active species (Scheme 9). It is conceivable that one of these steps may become
kinetically significant under some conditions, particularly if the water is
used both as a nucleophile and as a bridging ligand in binuclear enzymes.Scheme 9


### 3.3. Mechanisms

Although the metal-ion
requirement in MBLs catalysed hydrolysis ofβ-lactam antibiotics is still a
matter of debate, catalytic mechanisms have been proposed for both the mono-
and binuclear enzymes. The first catalytic mechanism suggested for BCII [25] (Scheme 10) was based on its crystal
structure at a resolution of 2.5  Å′, where only one
zinc-ion was bound to the enzyme, in the histidine site. The main features of
this mechanism are similar to those often proposed for zinc peptidases such as
carboxypeptidase A and thermolysin. Following substrate binding, the
zinc- bound water molecule, deprotonated by the Asp120 residue, attacks the
carbonyl centre with the formation of a negatively charged tetrahedral
intermediate, which is stabilised by its interactions with the metal-ion. The
Asp120 residue donates a proton to the nitrogen and C–N bond cleavage occurs,
followed by product dissociation from the enzyme active site. The main
disadvantage of this model is the unnecessary step of removing a proton from
the zinc-bound water as it is already ionised, although this may be required
during turnover.Scheme 10The pH-rate profile for the BCII catalysed hydrolysis of benzylpenicillin and
cephaloridine [49] was taken to indicate that the zinc-ion-bound
water has a low pKa of <5 and is therefore fully ionised at
neutral pH. Nucleophilic attack by the metal-bound hydroxide ion on the
carbonyl followed by a proton abstraction from the Asp120 gives a dianionic
tetrahedral intermediate (Scheme 11). It was suggested that the same
aspartate residue functions as proton donor to facilitate C–N bond fission, and
either step k2 or k3 could be rate limiting. A dianionic
intermediate assists β-lactam ring
opening and generates a carboxylate anion rather than the undissociated acid.Scheme 11Based on the crystal structure of
CcrA MBL fromBacteroides fragilis and on models of β-lactam substrates bound in the enzyme active site, a
mechanism of hydrolysis for the dizinc enzyme has been suggested to be
analogous to mechanisms proposed for other binuclear metallo-hydrolases [50]. In
this mechanism (Scheme 12), the
bridging hydroxide-ion is responsible for the nucleophilic attack which results
in a negatively charged intermediate stabilised by the oxyanion hole of the
enzyme. The apical water molecule bound to zinc is optimally positioned to
donate a proton to the leaving nitrogen, and the newly formed hydroxide-ion
moves to occupy the vacated Wat1 site, followed by product dissociation from the
enzyme active site. The original proposal involved zinc coordination to the β-lactam nitrogen, but this is unlikely because of its
relatively low electron density due to amide-resonance. However, nitrogen
binding to zinc is more likely once the tetrahedral intermediate is formed
because of its increased basicity [10].Scheme 12These studies used the unusualβ-lactam nitrocefin as substrate for CcrA, which,
because of the chromophoric product, allowed rapid scanning and single-wavelength
stopped-flow studies to reveal the accumulation, during turnover, of an enzyme
bound intermediate with an intense absorbance at 665 nm [51, 52]. This was
postulated to be an enzyme intermediate in which the leaving nitrogen atom is
not protonated during the cleavage of the C–N bond and remains negatively
charged (9). The anion is
stabilised by extensive conjugation with the dinitrostyryl substituent in the C3 position and by the zinc-ion in the second active site and so is an atypical
leaving group. Most amine anions are very unstable and are unlikely to be
expelled without N-protonation.(9)A similar intermediate has been
observed during the class B3 L1 enzyme-catalysed hydrolysis of nitrocefin, and,
as in the case of CcrA, its breakdown was rate-limiting [53]. However, stop-flow
studies of the tryptophan fluorescence revealed that the mechanism of
nitrocefin hydrolysis by binuclear metallo-β-lactamases may be unusual and
cleavage of the β-lactam amide bond is the rate determining step for the
breakdown of the majority of β-lactam substrates by the L1 enzyme [54].In contrast to these results, no
accumulation of an anionic intermediate could be detected in reactions
catalysed by BCII and the rate of
substrate depletion and product formation are similar [55]. However, for the BCII-catalysed
hydrolysis under cryoenzymological conditions, a slightly red-shifted
intermediate (440 nm) was observed, which was suggested to be a nitrocefin-like
tetrahedral intermediate [56].An unusual mechanism has been
proposed for subclass B2 metallo-β-lactamase CphA from Aeromonas hydrophila based on the crystal structures of the free
enzyme and of a reaction intermediate between the enzyme and the substrate
meropenem (Scheme 13) [57].
Nucleophilic attack is performed by a water molecule not coordinated to zinc, but activated by general base catalysis
by His118, followed by C–N bond fission which occurs prior to nitrogen protonation.
This proposal uses zinc as a Lewis acid to facilitate C–N bond fission
and to stabilise the leaving group—a mechanism proposed much earlier based on
model studies [10]—whereas most mechanisms use the zinc-ion to
act as a Lewis acid to stabilize oxyanion formation from the β-lactam carbonyl oxygen. Rearrangement of the proposed
intermediate amine anion, whose negatively charged nitrogen is stabilised by an
interaction with the zinc-ion, gives the bicyclic intermediate shown (Scheme 13), which is observed in the crystal structure, although
this rearrangement may not occur in the active site of the enzyme. A solvent molecule
replacing the water used in the nucleophilic attack protonates the nitrogen and
releases the final product.Scheme 13


### 3.4. Mutation Studies with MBLs

Many mutant enzymes from the three
subclasses of MBLs have been produced and characterised. In general, the
residues substituted have been the metal ligands, those providing the framework
for orienting the metal ligands or those thought to interact with the
substrates. For BCII, replacing
the histidine residues in the first binding site with serine resulted in
decrease of catalytic activity, although the affinity for zinc was not
significantly altered [58]. The decrease in activity appeared as due
to an increase in Km which was rationalised on the basis that, in absence of a His ligand, there is a higher degree of flexibility of the substrate
inside the catalytic site.The replacement of the Cys221 residue with  alanine
or serine in the active sites of BCII [58], CcrA
[59], and IMP-1 [60] gives a drastic decrease in the rate of
hydrolysis at low zinc concentrations, but the mutants are “reactivated” by the
addition of excess zinc, at least in case of BCII and IMP1. It was proposed that the
presence of Cys has a crucial importance for the catalytic activity of the
monozinc enzyme but not for the dinuclear species [58].Substituting the zinc 2 ligand Asp120
in BCII with Asn reduces the
catalytic activity by more than 100 fold, but does not impair the binding of
the second zinc-ion [58]. Although these results support the hypothesis that
Asp120 plays an important role in catalysis, the significant catalytic activity
of the mutants is inconsistent with Asp playing the role of a general base. The
same amino acid substitution in subclass B3 MBL enzyme L1 led to an average 10
fold decrease in catalytic activity in the binuclear mutant [61]. It is
interesting that for the hydrolysis of nitrocefin, this mutant shows an
unusually large kinetic solvent isotope effect (KSIE) of 5.36 compared with the
wild type (KSIE = 2.08) and other aspartate mutants: Asp120Cys and Asp120Ser
(KSIE <2). It is concluded that the Asp120 residue is essential for the
catalytic activity in the orientation of the bridging group of the L1 enzyme,
in the protonation step of breakdown of the tetrahedral intermediate, rather
than in its formation involving nucleophilic attack, as previously suggested
[62].The mutation of the second shell
ligand, Arg121Cys, in BCII gives
an enzyme with a lower affinity for the second metal-ion compared to the wild
type [58]. Moreover, the electronic spectrum of the dicobalt Arg121Cys BCII mutant is identical to that of the wild-type
enzyme indicating that the mutation has not altered the metal coordination
sphere. The catalytic activity of the mutant is only two fold lower
than that of the wild type for both the mono- and dinuclear forms of the
enzyme. The Cys104Arg mutation in CcrA led to a binuclear enzyme with significantly
decreased activity and which, unlike the wild type, can have one bound metal-ion
removed by a chelator [39]. Presteady state kinetics suggested a change in the
rate limiting step in the hydrolysis of nitrocefin, from nitrogen protonation
in the wild type, to C–N bond fission in the mutant, which showed a catalytic
rate constant similar to that of the dizinc BCII enzyme. The monozinc form of the mutant showed
a kcat value similar to that of the monozinc BCII,
for nitrocefin hydrolysis, with a further decrease of the rate of C–N bond
fission compared with the dizinc mutant, but with a similar protonation rate.
Based on these observations it was concluded that both the replacement of the
arginine residue and the introduction of the second metal-ion are evolutionary
tools for accelerating C–N bond fission in MBL catalysed hydrolysis of
β-lactams. However, an important role in lowering the energy barrier for
breaking the C–N bond must come from other enzyme structural rearrangements,
since the reciprocal, that is, the sole replacement of the arginine by a
cysteine residue and the insertion of a second zinc-ion in BCII, only marginally (two fold) improves the
catalytic activity [55].Another interesting mutagenesis study
has been carried out on class B2 CphA enzyme fromAeromonas hydrophila, which is a special case of an MBL, both
regarding its narrow substrate profile and its metal-ion requirement [63]. The
Asn116 residue in site one was mutated to a histidine in an attempt to create a
B1 type first binding site; this mutation changed the properties of the CphA
enzyme towards a broader substrate profile (characteristic to subclass B1), as
both penicillins and cephalosporins became significantly better substrates.
Moreover, the activity of the Asn116His mutant increased with increasing metal-ion
concentration for the hydrolysis of benzylpenicillin and cephaloridine, as
opposed to imipenem, where addition of zinc to both the mutant and the wild
type lead to noncompetitive inhibition. The Cys221Ser and Cys221Ala mutants
were seriously impaired in their ability to bind the first zinc-ion and were
nearly completely inactive indicating a major role for Cys221 in binding the
catalytic metal-ion.


### 3.5. Metal Ion Substitution

The exchange of the
spectroscopically silent zinc in zinc enzymes with probes, such as cobalt,
copper, and cadmium, enables the study of the metal interactions in the enzyme
active site with its ligands, substrates, and inhibitors of the metallo-enzyme
using techniques such as electronic spectroscopy, NMR, EPR, and perturbed
angular correlation (PAC) spectroscopy. The zinc of metalloβ-lactamases can be exchanged with cadmium, cobalt, and manganese to give
catalytically active enzymes. The
use of a combination of NMR and PAC spectroscopy to study cadmium binding to B. cereus MBL has revealed a rapid
intramolecular exchange of the metal between the two sites in the monocadmium
enzyme and negative cooperativity in metal binding [64]. The enzyme inhibitor
(R)-thiomandelate induces a very strong positive cooperativity for binding the
second cadmium cation [65].The metal-ion environment of cobalt-substituted metalloβ-lactamases has been studied by UV-vis
spectroscopy, NMR, and ESR. The
UV-vis spectra of cobalt-substituted class B1 metallo-β-lactamases CcrA from Bacteroidis fragilis [36, 66]
and BCII from Bacillus cereus [67, 68] show similar features with an
intense S to Co(II) ligand to metal charge transfer transition (LMCT) band at
340 and 348 nm and four characteristic d-d transition bands between 500 and 650 nm. The spectrum of Co-BCII in the
visible region is also very similar to that of Co(II) carbonic anhydrase at
alkaline pH [69], suggesting that the contributions from the His3 metal centre dominate this part of the spectrum of Co substituted class B1
MBLs.On
titration of monozinc BCII with
Co(II), the spectrum shows an LMCT band, but without the four ligand field
bands which suggests that zinc binds to the His3 site, while cobalt
preferentially occupies the Cys site in the hetero-substituted CoZn-enzyme [67].
A similar discrimination between metals in occupying the two binding sites in BCII was noted for the CdZn-enzyme, where zinc also
binds in the His3 site and Cd to the Cys site [64]. Studies of
cobalt binding to apo BCII, using HEPES as a buffer and no added sodium
chloride, showed that increasing the ratio Co(II)/enzyme above one resulted in
a decrease of the charge transfer band at 344 nm and the appearance of an
additional charge transfer band at 383 nm. This has been explained by the
binding of a second cobalt ion to the monocobalt BCII species and the existence of two different Co(II) LMCT band positions, one for
the mononuclear (344 nm) and one for the binuclear enzyme (383 nm); this latter
band disappears upon addition of sodium chloride, accompanied by an increase in
absorbance at 344 nm, which led to the suggestion that chloride hinders the
binding of a second cobalt ion [34]. More recent data supported by NMR and EPRevidence have shown that the band at 383 nm
corresponds to a third, more weakly bound cobalt ion that perturbs the 344 nm
signal (the Cys site), without affecting the spectral features of the histidine
site [70].Low-temperature
EPR spectra of
metallo-β-lactamases indicate the presence of high spin, but not coupled,
Co(II) ions in a rhombically distorted pentacoordination sphere in Co-substituted
BCII [68], a penta/hexacoordination environment in Co-substituted CcrA enzyme
[36], a tetracoordination environment for the first cobalt ion, and  penta/hexacoordination sphere for the second
cobalt ion in Co-substituted ImiS [71]. For this latter enzyme, it has been
shown by EPR that the second
cobalt ion is magnetically isolated, suggesting a distance of more than 7 Å
from the first bound cobalt ion. Furthermore, NMR titration of the monocobalt
ImiS enzyme with Co(II) indicated that the second cobalt ion is not bound to
histidine, as the newly observed resonances are not solvent exchangeable; these
findings were taken to indicate that the Zn1 site has no catalytic or metal
binding role in ImiS, that Zn2 site binds the metal-ion that is required for
catalysis, and a remote, lower-affinity, metal binding site is responsible for
the noncompetitive inhibition of the enzyme [72]. From the
mechanistic point of view, it has been shown by time dependent UV-vis
spectroscopy and fluorescence quenching, using nitrocefin as a substrate, that
Co-substituted L1, a class B3 metallo-β-lactamase, probably utilises a reaction
mechanism similar to that
of the native zinc
enzyme [54, 73]. Rapid-freeze-quench (RFQ) EPR shows that a short-lived intermediate is a metal-bound species, and the role of
the metal-ion in catalysis is similar for nitrocefin, cephalothin, meropenem,
and benzylpenicillin [73].The
kinetics and mechanism of hydrolysis of theB.
cereus (BcII) metallo-β-lactamase substituted with various metal-ions have been
investigated to determine the role of the active site metal-ion [74]. The pH
and metal-ion dependence of kcat and kcat/Km for the cobalt-substituted BcII catalysed hydrolysis of cefoxitin,
cephaloridine, and cephalexin indicate that an enzyme residue of apparent pKa 6.3 is required in its deprotonated form for metal-ion binding and catalysis.
The kcat/Km for cefoxitin and cephalexin hydrolysis with
cadmium-substituted BcII is dependent on two ionising groups on the enzyme: one
of pKa1 8.7, required in its deprotonated form, and the other of pKa2 9.3, required in its protonated form for activity. The identity of these
residues was determined from the pH-dependence of the competitive inhibition
constant, Ki, of the Cd BcII by L-captopril which showed that the pKa1 of 8.7 corresponds to the cadmium-bound water. For the manganese-substituted
BcII, the pH-dependence of kcat/Km for the hydrolysis of β-lactam
antibiotics similarly indicated the importance of two catalytic groups: one of
pKa1 8.5 which needs to be deprotonated and the other of pKa2 9.4 which needs to be protonated for catalysis; the pKa1 was
assigned to the manganese-bound water [74]. Interestingly, the metal-substituted
enzymes have similar or higher catalytic activities compared with the native zinc
enzyme, albeit at pHs above 7 and, for the Co enzyme, at all pHs (Figure 1). With
cefoxitin, a very poor substrate for Zn BcII, both kcat and kcat/Km increase with increasing pKa of the metal-bound water, in the order
Zn<Co<Mn<Cd. A higher pKa for the metal-bound water for
cadmium and manganese BCII leads
to more reactive enzymes than the native zinc BcII, suggesting that the role of
the metal-ion is predominantly to provide the nucleophilic hydroxide, rather
than to act as a Lewis acid to polarise the carbonyl group and stabilise the
oxyanion tetrahedral intermediate [74].Figure 1
Plot of logkcat/Km for (▪) ZnBCII-, (•) CoBCII-,  (▴) CdBCII-, and (x) MnBCII-catalysed
hydrolysis of cephalexin.Given the relatively weak binding of the second
zinc in theB. cereus (BcII)
MBL and the fact that an important ligand
holding the metal-ion to the protein is the bridging water which is consumed during the catalytic cycle of
hydrolysis, it is possible that the second metal-ion could be lost during
turnover. In fact, the kinetics of the hydrolysis of benzylpenicillin
catalysed by the cobalt substituted β-lactamase from B. cereus (BcII) are biphasic with an
initial burst of product formation followed by a steady-state rate of hydolysis
[75]. This was interpreted as being due to a branched kinetic pathway with two
enzyme intermediate species, ES1 and ES2, which have
different metal: enzyme stoichiometries. ES1 is a dicobalt enzyme
intermediate and is catalytic, but is slowly losing one bound cobalt ion during
turnover via the branching route, to give the mononuclear and inactive enzyme
intermediate ES2. The dependence of enzyme activity on pH and metal-ion
concentration indicates that only the dicobalt enzyme is catalytically active. The
monocobalt enzyme species, formed during the catalytic cycle, is virtually
inactive and requires the association of another cobalt ion for turnover. The
dicobalt enzyme intermediate is responsible for the direct catalytic route,
which is pH-independent between 5.5 and 9.5. The inactivation pathway of
metal-ion dissociation occurs by both an acid catalysed and a pH-independent reaction,
which is dependent on the presence of an enzyme residue of pKa 8.9
in its protonated form and shows a large kinetic solvent isotope effect (H2O/D2O)
of 5.2, indicative of a rate limiting proton transfer [75]. The pseudo
first-order rate constant to regenerate the dicobalt β-lactamase from
the monocobalt enzyme intermediate has a first-order dependence on cobalt ion
concentration. This unusual behaviour is attributed to an intrinsic property of
metallo hydrolytic enzymes that depend on a metal-bound water both as a ligand
for the second metal-ion and as the nucleophile which is consumed during
hydrolysis of the substrate and so has to be replaced to maintain the catalytic
cycle [75, 76].There remain questions regarding the necessity of the binuclear metal
centre itself: (i) are metallo-enzyme species responsible for the physiological
activity, the binuclear one or the mononuclear one, or are they interchangeable
under certain conditions? and if so, (ii) are these changes relevant to any
other functions, apart from catalysis, such as regulation of activity? It
does seem intrinsically odd in the binuclear enzyme that the bridging
hydroxide-ion is used as a nucleophile because of its presumed weak basicity.
Hydroxide-ion has three lone pairs, two of which are taken up by metal-ion
coordination and one of which is required for nucleophilic attack and covalent
bond formation. If this bridging hydroxide-ion is used as the attacking
nucleophile is it activated by general base catalysis so that concerted proton
removal generates a dianionic tetrahedral intermediate?


## 3.1. The Number of Zinc-Ions

A major problem with
understanding the mechanism of MBLs is the number of zinc-ions required for
catalysis, which has been addressed by studying MBLs under both equilibrium and
kinetic conditions. The crystal structures of several MBLs have been determined
by X-ray diffraction and they all  show a
similarαβ/βα sandwich fold, which was first seen with MBLs
[25], but since recognised in other enzymes, such as glyoxalase II, aryl
sulfatase, and cAMP phosphodiesterase, which have
now become members of the MBL fold superfamily [26]. The active site of MBLs is
situated at the bottom of a wide shallow groove between two β-sheets and has two potential zinc-ion binding sites at
the active site, often referred to as sites 1 and 2 [27–29]. The zinc ligands
in the two sites are not the same and are not fully conserved between the
different MBLs. Table 1 shows
the enzyme residues involved in zinc coordination in the two binding sites in
the subclasses B1, B2, and B3 [26].Table 1
The zinc
ligands in class Bβ-lactamases.
SubclassZn1 ligandsZn2 ligandsB1His116His118His196Asp120Cys221His263B2Asn116His118His196Asp120Cys221His263B3His/Gln116His118His196Asp120His121His263In the subclass B1, such as
theBacillus cereus enzyme BCII, the zinc in site 1 (the histidine site or His3 site) is tetracoordinated by the imidazole groups of three histidine
residues (116, 118, and 196) and a water molecule, Wat1.  In site 2 (or the Cys site) the metal is
pentacoordinated by His263, Asp120, Cys221, and one water molecule; the fifth
ligand at site 2 is carbonate or water, often referred to as the apical water,
or Wat2 [27, 28, 30], although this is missing in structures with
inhibitors bound [31]. The two metal-ions are relatively close to each other,
but the distance between them varies from 3.4 to 4.4 Å  in different structures
of the BCII and CcrA enzymes.  Several structures of the CcrA enzyme show a
bridging water ligand between the two metals, which is thought to exist as a
hydroxide-ion [32]. In a structure of BCII determined at pH 7.5 that contains two zinc-ions there is also a similar bridging water molecule, but in structures of this enzyme at lower pH this
solvent molecule is strongly associated to the zinc in site 1 [30].The two conserved zinc
binding sites in MBLs have different metal-ion affinities. For example, the BCII enzyme fromBacillus cereus has very different dissociation constants for the
two metal binding sites. The first crystal structure, obtained at low pH [25], indicated
only one zinc-ion bound to the histidine site, but equilibrium dialysis studies
showed two binding events, with a dissociation constant of 0.3 μM for the first
zinc-ion (Kmono) and 3 μM for the second zinc-ion (Kbi)
[33]. Later metal binding studies by fluorescence spectroscopy using a
chromophoric chelator reported a Kmono of 0.62 nM and a Kbi of 1.5 μM [34]. As the conditions of the experiments were similar for the two
studies, the large discrepancy in Kmono may be due to the fact that
different strains of BCII enzyme
from Bacillus cereus were used. The dissociation
constants Kmono and Kbi have been determined from the
steady state rates of hydrolysis of imipenem in the presence of EDTA as a metal-ion
buffer at different zinc concentrations and Kmono was found to
decrease significantly, from nM to pM, in presence of substrate, whereas Kbi decreased only by two fold [35]. This led to the suggestion that the monozinc
enzyme is responsible for the catalytic activity under physiological
conditions, where the concentration of the “free" Zn2+ is in the pM or
even fM region. For most substrates, the reported catalytic activity of the
monozinc BCII was about two fold
lower than that of the binuclear enzyme. Conversely, the other class
B1 enzyme, CcrA
from Bacteroides fragilis, binds both
zinc-ions very tightly [36].  Despite the
very close similarity with BCII,
CcrA has much higher affinity for the second zinc-ion, probably due to the fact
that in CcrA, a cysteine residue replaces the positively charged arginine 121
found in BCII.  However, replacing Arg 121 in BCII by a Cys shows no increase in the affinity for
the second zinc-ion [37].Early kinetic studies of CcrA
led to the proposal thatboth the
mono and the dinuclear forms of the enzyme were catalytically active, with
slightly different activities, at physiological pH [38]. However, later studies showed that only the
dinuclear species was active and that the previously observed “monozinc” CcrA
was a mixture of the dizinc and the apo (metal free) enzyme [39]. Class
B2 metallo β-lactamases are catalytically active with one bound zinc-ion and
the binding of the second zinc-ion inhibits the enzyme noncompetitively, with a
Ki of 50 μM [40]. The dissociation constant of the first zinc-ion was
found to be 7.0 and 1.2 pM in the absence and presence of substrate (imipenem),
respectively [35].  Subclass B3 MBLs, L1,
and FEZ-1 bind both metal-ions tightly, the dissociation constants are in the
nM region, and are fully catalytically active in binuclear form [23, 41].


## 3.2. The Catalytic Role of Zinc

Zinc is an essential trace element
and the second most abundant transition metal found in living sytems. Its role
in catalysis is related to its ability to participate in tight, but readily exchangeable
ligand binding and its exceptional flexibility of its coordination number and geometry
[42]. In addition, zinc shows no redox properties and this facilitates its
evolution in living systems without the risk of oxidative damage. Finally, its intermediate
hard-soft behaviour allows it to bind a variety of atoms, as seen, for example,
in the second binding site of class B1 MBLs which involve nitrogen, oxygen, and
sulphur as ligands. The Lewis acidity, flexible geometry, and coordination
number and the lack of redox properties make zinc an ideal metal cofactor for many
enzymes. The small energy difference between 4, 5 or 6 coordination geometries
and the rapid exchange of the kinetically labile zinc-bound water molecule are
important features in all zinc hydrolases including MBLs.MBLs
are a subclass of metallo-proteases and many mechanistic considerations are
applicable to both groups. There are many potential mechanistic roles for the
metal-ion in metallo-proteases and MBLs, although they may well vary from
enzyme to enzyme [42, 43]. It is commonly suggested that the metal-ion acts as
a Lewis acid by coordination to the peptide carbonyl oxygen giving a more
electron deficient carbonyl carbon which facilitates nucleophilic attack. The
metal-ion thus stabilises the negative charge developed on the carbonyl oxygen
of the tetrahedral intermediate anion (Scheme
3). Many metallo-proteases have a water molecule directly coordinated to
the metal-ion which may act as the nucleophile to attack the carbonyl
carbon.  Here, the role of the metal-ion
is to lower the pKa of the coordinated water so that the
concentration of metal-bound hydroxide ion is increased relative to bulk
solvent hydroxide-ion at neutral pH and, furthermore, although this coordinated
hydroxide-ion is different in nature than the simple solvated ion, it is a
better nucleophile than water (Scheme 4).
These two steps shown in Schemes 3 and 4 relate to
formation of the tetrahedral intermediate and although little attention is
normally given to the mechanism of the breakdown of the tetrahedral
intermediate, C–N bond fission is the most energetically difficult process in
peptide hydrolysis. This could be facilitated by direct coordination of the
departing amine nitrogen to the metal-ion (Scheme 5) which is, in
fact, the mechanism adopted for the simple zinc-ion-catalysed hydrolysis of
penicillin [10]. Alternatively, a metal-bound water could act as a general acid
catalyst protonating the amine nitrogen leaving group to facilitate C–N bond
fission (Scheme 6). Despite
intense mechanistic studies, the detailed roles of the metal-ion in
metallo-proteases and MBLs remain controversial and distinguishing between the
relative importance of the possible roles for zinc is complex.Scheme 3Scheme 4Scheme 5Scheme 6The effective positive charge on the zinc-ion depends on the number and nature of
its ligands and for those with an ionisable hydrogen, zinc lowers theirpKa and the ionised ligand obviously is better at neutralising the positive charge
density on the metal. The pKa of water is 15.7, but when bound to zinc
in aqueous solution it is lowered to 9.5, but is there evolutionary pressure to
lower the pKa of the zinc-bound water in an enzyme even further? This
could be achieved by replacing
say a negatively charged carboxylate ligand by a  neutral histidine. For example, in the
opposite direction, changing the histidine bound to zinc in carbonic anhydrase to
aspartate increases the pKa of the zinc-bound water from 6.8 to ≥ 9.6
[45]. Whether it is better to have a higher or lower pKa metal-bound
water for a faster reaction and more efficient catalysis depends on the role of
the zinc-ion. A low pKa implies a more electron deficient metal-ion
centre which would give a better Lewis acid to stabilise the negative charge
developed on the oxyanion of the tetrahedral intermediate. Conversely, a high
pKa for the metal-bound water implies a weaker Lewis acid and so the
zinc-ion will be less efficient at stabilising the tetrahedral intermediate. A
low pKa for the metal-bound water implies that the hydroxide-ion resulting
from ionisation is more “tightly bound” and stabilised which, although it
becomes the dominant species even at low pH, corresponds to a more weakly
nucleophilic hydroxide-ion. For example, if the pKa of the zinc-bound
water is about 5 then the nucleophilicity of the metal bound hydroxide-ion is
only similar to that of a carboxylate anion. If a major role of the metal-ion
is to provide a better nucleophile than water then the net effect depends on
the relative importance of concentration and the dependence of the rate upon
nucleophilicity. If the pKa is “too high”, metal-coordinated water
will be the dominant species over the desired pH range but deprotonation will
give a more nucleophilic metal-bound hydroxide. How reactivity changes with
changing pKa and pH will depend on the susceptibility of the rate of
reaction to the basicity of the nucleophile—the hydroxide-ion bound to the metal—as indicated by the Bronsted βnuc value.Sometimes it is suggested that part of the enzyme mechanism involves general base
catalysis to remove the proton from the zinc-bound water but this only becomes
necessary if thepKa is little changed from that of bulk water. A
problem with the commonly accepted mechanism for metallo-proteases of rate-limiting
deprotonation of zinc-bound water concerted with nucleophilic attack is the pKa of this water.  Coordination to zinc(II) generally
lowers the pKa of water to 5–9 depending on the number and type of
other ligands.  The concentration of
zinc-bound hydroxide ion is therefore quite high over the normal pH range
studied. There is little or no catalytic advantage in having a general base
to remove a proton in this pre-equilibrium step!  Even if the pKa of the
zinc(II)-bound water is about 9 then 10% of the species already exists in the
fully deprotonated form at pH 8 and 1% at pH 7.
Presumably, the deprotonated form is a much better nucleophile than the
species which is only partially deprotonated and there would be no catalytic
advantage of the general base-catalysed mechanism.In
aqueous solution, zinc is coordinated to six water molecules and thepKa of the zinc-bound water is 9.5. However, within a protein this pKa can be changed significantly because of the environment and directly bonded
ligands to the metal—for example, the zinc of B. cereusβ-lactamase is coordinated to three protein ligands in
the zinc1 site—His 116, His 118, and His 196 and a water molecule [30]. The
reduced coordination number of four in the zinc1 site of β-lactamase reduces the pKa to less
than 6. In principle, one or more of the imidazole residues could be ionised,
the pKa for proton loss from imidazole is similar to that of water (about
14). However, NMR evidence suggests that the imidazole of the bound histidines
are neutral [46, 47] and the most likely group to ionise is, therefore, the bound
water (Scheme 7).Scheme 7One
way to investigate the role of the zinc in catalysis is to modify thepKa of the zinc-bound water by changing the ligands or the metal-ion. If the
activity of the resulting enzyme increases as the pKa of the zinc-bound water ligand decreases, this would suggest that a high positive charge density
on the metal-ion facilitates catalysis and that the zinc coordination to the
carbonyl oxygen and stabilisation of the negative charge developed on this
oxygen following nucleophilic attack is important. A lower pKa of
zinc-bound water indicates a more electrophilic zinc which is better at
stabilising negative charge giving rise to a better catalyst. Stabilisation of
the intermediate anion must be more important
than the nucleophilicity of the zinc hydroxide.  Conversely, if activity increases with
increasing   pKa of the zinc-bound water, that is,
increasing basicity of the of the zinc-bound hydroxide ion, then this would
indicate a greater role for the metal-ion in controlling the nucleophilicity of
the hydroxide-ion compared with its role in stabilising the negative charge
development on the carbonyl oxygen.Thedual role of zinc in metallo-proteases, that is, acting as a Lewis
acid in polarising the carbonyl bond and as source of nucleophilic hydroxide,
requires formally the formation of an apparently strained four-membered ring (Scheme 8). However, this coordination occurs in the
bidentate complexes of zinc with carboxylate anions. Monodentate complexes
between zinc and a carboxylate anion exist predominantly as the syn stereoisomer due to the more basic syn lone pair and favourable opposition
of the C=O and O–Zn dipoles. The coordination adopted for the syn conformation is similar to a
bidentate one which is favoured by electrostatic interactions [48].Scheme 8One
final point to consider for the hydrolysis ofβ-lactams by metallo-β-lactamases is that if the zinc bound hydroxide-ion
is the nucleophile then it is consumed during turnover. Consequently, regeneration of the catalyst requires the
coordination of a new water molecule to the active site zinc. For example, if
product release occurs through displacement by water or is introduced in a
separate step following product release, deprotonation must occur to generate a catalytically active species (Scheme 9). It is conceivable that one of these steps may become
kinetically significant under some conditions, particularly if the water is
used both as a nucleophile and as a bridging ligand in binuclear enzymes.Scheme 9


## 3.3. Mechanisms

Although the metal-ion
requirement in MBLs catalysed hydrolysis ofβ-lactam antibiotics is still a
matter of debate, catalytic mechanisms have been proposed for both the mono-
and binuclear enzymes. The first catalytic mechanism suggested for BCII [25] (Scheme 10) was based on its crystal
structure at a resolution of 2.5  Å′, where only one
zinc-ion was bound to the enzyme, in the histidine site. The main features of
this mechanism are similar to those often proposed for zinc peptidases such as
carboxypeptidase A and thermolysin. Following substrate binding, the
zinc- bound water molecule, deprotonated by the Asp120 residue, attacks the
carbonyl centre with the formation of a negatively charged tetrahedral
intermediate, which is stabilised by its interactions with the metal-ion. The
Asp120 residue donates a proton to the nitrogen and C–N bond cleavage occurs,
followed by product dissociation from the enzyme active site. The main
disadvantage of this model is the unnecessary step of removing a proton from
the zinc-bound water as it is already ionised, although this may be required
during turnover.Scheme 10The pH-rate profile for the BCII catalysed hydrolysis of benzylpenicillin and
cephaloridine [49] was taken to indicate that the zinc-ion-bound
water has a low pKa of <5 and is therefore fully ionised at
neutral pH. Nucleophilic attack by the metal-bound hydroxide ion on the
carbonyl followed by a proton abstraction from the Asp120 gives a dianionic
tetrahedral intermediate (Scheme 11). It was suggested that the same
aspartate residue functions as proton donor to facilitate C–N bond fission, and
either step k2 or k3 could be rate limiting. A dianionic
intermediate assists β-lactam ring
opening and generates a carboxylate anion rather than the undissociated acid.Scheme 11Based on the crystal structure of
CcrA MBL fromBacteroides fragilis and on models of β-lactam substrates bound in the enzyme active site, a
mechanism of hydrolysis for the dizinc enzyme has been suggested to be
analogous to mechanisms proposed for other binuclear metallo-hydrolases [50]. In
this mechanism (Scheme 12), the
bridging hydroxide-ion is responsible for the nucleophilic attack which results
in a negatively charged intermediate stabilised by the oxyanion hole of the
enzyme. The apical water molecule bound to zinc is optimally positioned to
donate a proton to the leaving nitrogen, and the newly formed hydroxide-ion
moves to occupy the vacated Wat1 site, followed by product dissociation from the
enzyme active site. The original proposal involved zinc coordination to the β-lactam nitrogen, but this is unlikely because of its
relatively low electron density due to amide-resonance. However, nitrogen
binding to zinc is more likely once the tetrahedral intermediate is formed
because of its increased basicity [10].Scheme 12These studies used the unusualβ-lactam nitrocefin as substrate for CcrA, which,
because of the chromophoric product, allowed rapid scanning and single-wavelength
stopped-flow studies to reveal the accumulation, during turnover, of an enzyme
bound intermediate with an intense absorbance at 665 nm [51, 52]. This was
postulated to be an enzyme intermediate in which the leaving nitrogen atom is
not protonated during the cleavage of the C–N bond and remains negatively
charged (9). The anion is
stabilised by extensive conjugation with the dinitrostyryl substituent in the C3 position and by the zinc-ion in the second active site and so is an atypical
leaving group. Most amine anions are very unstable and are unlikely to be
expelled without N-protonation.(9)A similar intermediate has been
observed during the class B3 L1 enzyme-catalysed hydrolysis of nitrocefin, and,
as in the case of CcrA, its breakdown was rate-limiting [53]. However, stop-flow
studies of the tryptophan fluorescence revealed that the mechanism of
nitrocefin hydrolysis by binuclear metallo-β-lactamases may be unusual and
cleavage of the β-lactam amide bond is the rate determining step for the
breakdown of the majority of β-lactam substrates by the L1 enzyme [54].In contrast to these results, no
accumulation of an anionic intermediate could be detected in reactions
catalysed by BCII and the rate of
substrate depletion and product formation are similar [55]. However, for the BCII-catalysed
hydrolysis under cryoenzymological conditions, a slightly red-shifted
intermediate (440 nm) was observed, which was suggested to be a nitrocefin-like
tetrahedral intermediate [56].An unusual mechanism has been
proposed for subclass B2 metallo-β-lactamase CphA from Aeromonas hydrophila based on the crystal structures of the free
enzyme and of a reaction intermediate between the enzyme and the substrate
meropenem (Scheme 13) [57].
Nucleophilic attack is performed by a water molecule not coordinated to zinc, but activated by general base catalysis
by His118, followed by C–N bond fission which occurs prior to nitrogen protonation.
This proposal uses zinc as a Lewis acid to facilitate C–N bond fission
and to stabilise the leaving group—a mechanism proposed much earlier based on
model studies [10]—whereas most mechanisms use the zinc-ion to
act as a Lewis acid to stabilize oxyanion formation from the β-lactam carbonyl oxygen. Rearrangement of the proposed
intermediate amine anion, whose negatively charged nitrogen is stabilised by an
interaction with the zinc-ion, gives the bicyclic intermediate shown (Scheme 13), which is observed in the crystal structure, although
this rearrangement may not occur in the active site of the enzyme. A solvent molecule
replacing the water used in the nucleophilic attack protonates the nitrogen and
releases the final product.Scheme 13


## 3.4. Mutation Studies with MBLs

Many mutant enzymes from the three
subclasses of MBLs have been produced and characterised. In general, the
residues substituted have been the metal ligands, those providing the framework
for orienting the metal ligands or those thought to interact with the
substrates. For BCII, replacing
the histidine residues in the first binding site with serine resulted in
decrease of catalytic activity, although the affinity for zinc was not
significantly altered [58]. The decrease in activity appeared as due
to an increase in Km which was rationalised on the basis that, in absence of a His ligand, there is a higher degree of flexibility of the substrate
inside the catalytic site.The replacement of the Cys221 residue with  alanine
or serine in the active sites of BCII [58], CcrA
[59], and IMP-1 [60] gives a drastic decrease in the rate of
hydrolysis at low zinc concentrations, but the mutants are “reactivated” by the
addition of excess zinc, at least in case of BCII and IMP1. It was proposed that the
presence of Cys has a crucial importance for the catalytic activity of the
monozinc enzyme but not for the dinuclear species [58].Substituting the zinc 2 ligand Asp120
in BCII with Asn reduces the
catalytic activity by more than 100 fold, but does not impair the binding of
the second zinc-ion [58]. Although these results support the hypothesis that
Asp120 plays an important role in catalysis, the significant catalytic activity
of the mutants is inconsistent with Asp playing the role of a general base. The
same amino acid substitution in subclass B3 MBL enzyme L1 led to an average 10
fold decrease in catalytic activity in the binuclear mutant [61]. It is
interesting that for the hydrolysis of nitrocefin, this mutant shows an
unusually large kinetic solvent isotope effect (KSIE) of 5.36 compared with the
wild type (KSIE = 2.08) and other aspartate mutants: Asp120Cys and Asp120Ser
(KSIE <2). It is concluded that the Asp120 residue is essential for the
catalytic activity in the orientation of the bridging group of the L1 enzyme,
in the protonation step of breakdown of the tetrahedral intermediate, rather
than in its formation involving nucleophilic attack, as previously suggested
[62].The mutation of the second shell
ligand, Arg121Cys, in BCII gives
an enzyme with a lower affinity for the second metal-ion compared to the wild
type [58]. Moreover, the electronic spectrum of the dicobalt Arg121Cys BCII mutant is identical to that of the wild-type
enzyme indicating that the mutation has not altered the metal coordination
sphere. The catalytic activity of the mutant is only two fold lower
than that of the wild type for both the mono- and dinuclear forms of the
enzyme. The Cys104Arg mutation in CcrA led to a binuclear enzyme with significantly
decreased activity and which, unlike the wild type, can have one bound metal-ion
removed by a chelator [39]. Presteady state kinetics suggested a change in the
rate limiting step in the hydrolysis of nitrocefin, from nitrogen protonation
in the wild type, to C–N bond fission in the mutant, which showed a catalytic
rate constant similar to that of the dizinc BCII enzyme. The monozinc form of the mutant showed
a kcat value similar to that of the monozinc BCII,
for nitrocefin hydrolysis, with a further decrease of the rate of C–N bond
fission compared with the dizinc mutant, but with a similar protonation rate.
Based on these observations it was concluded that both the replacement of the
arginine residue and the introduction of the second metal-ion are evolutionary
tools for accelerating C–N bond fission in MBL catalysed hydrolysis of
β-lactams. However, an important role in lowering the energy barrier for
breaking the C–N bond must come from other enzyme structural rearrangements,
since the reciprocal, that is, the sole replacement of the arginine by a
cysteine residue and the insertion of a second zinc-ion in BCII, only marginally (two fold) improves the
catalytic activity [55].Another interesting mutagenesis study
has been carried out on class B2 CphA enzyme fromAeromonas hydrophila, which is a special case of an MBL, both
regarding its narrow substrate profile and its metal-ion requirement [63]. The
Asn116 residue in site one was mutated to a histidine in an attempt to create a
B1 type first binding site; this mutation changed the properties of the CphA
enzyme towards a broader substrate profile (characteristic to subclass B1), as
both penicillins and cephalosporins became significantly better substrates.
Moreover, the activity of the Asn116His mutant increased with increasing metal-ion
concentration for the hydrolysis of benzylpenicillin and cephaloridine, as
opposed to imipenem, where addition of zinc to both the mutant and the wild
type lead to noncompetitive inhibition. The Cys221Ser and Cys221Ala mutants
were seriously impaired in their ability to bind the first zinc-ion and were
nearly completely inactive indicating a major role for Cys221 in binding the
catalytic metal-ion.


## 3.5. Metal Ion Substitution

The exchange of the
spectroscopically silent zinc in zinc enzymes with probes, such as cobalt,
copper, and cadmium, enables the study of the metal interactions in the enzyme
active site with its ligands, substrates, and inhibitors of the metallo-enzyme
using techniques such as electronic spectroscopy, NMR, EPR, and perturbed
angular correlation (PAC) spectroscopy. The zinc of metalloβ-lactamases can be exchanged with cadmium, cobalt, and manganese to give
catalytically active enzymes. The
use of a combination of NMR and PAC spectroscopy to study cadmium binding to B. cereus MBL has revealed a rapid
intramolecular exchange of the metal between the two sites in the monocadmium
enzyme and negative cooperativity in metal binding [64]. The enzyme inhibitor
(R)-thiomandelate induces a very strong positive cooperativity for binding the
second cadmium cation [65].The metal-ion environment of cobalt-substituted metalloβ-lactamases has been studied by UV-vis
spectroscopy, NMR, and ESR. The
UV-vis spectra of cobalt-substituted class B1 metallo-β-lactamases CcrA from Bacteroidis fragilis [36, 66]
and BCII from Bacillus cereus [67, 68] show similar features with an
intense S to Co(II) ligand to metal charge transfer transition (LMCT) band at
340 and 348 nm and four characteristic d-d transition bands between 500 and 650 nm. The spectrum of Co-BCII in the
visible region is also very similar to that of Co(II) carbonic anhydrase at
alkaline pH [69], suggesting that the contributions from the His3 metal centre dominate this part of the spectrum of Co substituted class B1
MBLs.On
titration of monozinc BCII with
Co(II), the spectrum shows an LMCT band, but without the four ligand field
bands which suggests that zinc binds to the His3 site, while cobalt
preferentially occupies the Cys site in the hetero-substituted CoZn-enzyme [67].
A similar discrimination between metals in occupying the two binding sites in BCII was noted for the CdZn-enzyme, where zinc also
binds in the His3 site and Cd to the Cys site [64]. Studies of
cobalt binding to apo BCII, using HEPES as a buffer and no added sodium
chloride, showed that increasing the ratio Co(II)/enzyme above one resulted in
a decrease of the charge transfer band at 344 nm and the appearance of an
additional charge transfer band at 383 nm. This has been explained by the
binding of a second cobalt ion to the monocobalt BCII species and the existence of two different Co(II) LMCT band positions, one for
the mononuclear (344 nm) and one for the binuclear enzyme (383 nm); this latter
band disappears upon addition of sodium chloride, accompanied by an increase in
absorbance at 344 nm, which led to the suggestion that chloride hinders the
binding of a second cobalt ion [34]. More recent data supported by NMR and EPRevidence have shown that the band at 383 nm
corresponds to a third, more weakly bound cobalt ion that perturbs the 344 nm
signal (the Cys site), without affecting the spectral features of the histidine
site [70].Low-temperature
EPR spectra of
metallo-β-lactamases indicate the presence of high spin, but not coupled,
Co(II) ions in a rhombically distorted pentacoordination sphere in Co-substituted
BCII [68], a penta/hexacoordination environment in Co-substituted CcrA enzyme
[36], a tetracoordination environment for the first cobalt ion, and  penta/hexacoordination sphere for the second
cobalt ion in Co-substituted ImiS [71]. For this latter enzyme, it has been
shown by EPR that the second
cobalt ion is magnetically isolated, suggesting a distance of more than 7 Å
from the first bound cobalt ion. Furthermore, NMR titration of the monocobalt
ImiS enzyme with Co(II) indicated that the second cobalt ion is not bound to
histidine, as the newly observed resonances are not solvent exchangeable; these
findings were taken to indicate that the Zn1 site has no catalytic or metal
binding role in ImiS, that Zn2 site binds the metal-ion that is required for
catalysis, and a remote, lower-affinity, metal binding site is responsible for
the noncompetitive inhibition of the enzyme [72]. From the
mechanistic point of view, it has been shown by time dependent UV-vis
spectroscopy and fluorescence quenching, using nitrocefin as a substrate, that
Co-substituted L1, a class B3 metallo-β-lactamase, probably utilises a reaction
mechanism similar to that
of the native zinc
enzyme [54, 73]. Rapid-freeze-quench (RFQ) EPR shows that a short-lived intermediate is a metal-bound species, and the role of
the metal-ion in catalysis is similar for nitrocefin, cephalothin, meropenem,
and benzylpenicillin [73].The
kinetics and mechanism of hydrolysis of theB.
cereus (BcII) metallo-β-lactamase substituted with various metal-ions have been
investigated to determine the role of the active site metal-ion [74]. The pH
and metal-ion dependence of kcat and kcat/Km for the cobalt-substituted BcII catalysed hydrolysis of cefoxitin,
cephaloridine, and cephalexin indicate that an enzyme residue of apparent pKa 6.3 is required in its deprotonated form for metal-ion binding and catalysis.
The kcat/Km for cefoxitin and cephalexin hydrolysis with
cadmium-substituted BcII is dependent on two ionising groups on the enzyme: one
of pKa1 8.7, required in its deprotonated form, and the other of pKa2 9.3, required in its protonated form for activity. The identity of these
residues was determined from the pH-dependence of the competitive inhibition
constant, Ki, of the Cd BcII by L-captopril which showed that the pKa1 of 8.7 corresponds to the cadmium-bound water. For the manganese-substituted
BcII, the pH-dependence of kcat/Km for the hydrolysis of β-lactam
antibiotics similarly indicated the importance of two catalytic groups: one of
pKa1 8.5 which needs to be deprotonated and the other of pKa2 9.4 which needs to be protonated for catalysis; the pKa1 was
assigned to the manganese-bound water [74]. Interestingly, the metal-substituted
enzymes have similar or higher catalytic activities compared with the native zinc
enzyme, albeit at pHs above 7 and, for the Co enzyme, at all pHs (Figure 1). With
cefoxitin, a very poor substrate for Zn BcII, both kcat and kcat/Km increase with increasing pKa of the metal-bound water, in the order
Zn<Co<Mn<Cd. A higher pKa for the metal-bound water for
cadmium and manganese BCII leads
to more reactive enzymes than the native zinc BcII, suggesting that the role of
the metal-ion is predominantly to provide the nucleophilic hydroxide, rather
than to act as a Lewis acid to polarise the carbonyl group and stabilise the
oxyanion tetrahedral intermediate [74].Figure 1
Plot of logkcat/Km for (▪) ZnBCII-, (•) CoBCII-,  (▴) CdBCII-, and (x) MnBCII-catalysed
hydrolysis of cephalexin.Given the relatively weak binding of the second
zinc in theB. cereus (BcII)
MBL and the fact that an important ligand
holding the metal-ion to the protein is the bridging water which is consumed during the catalytic cycle of
hydrolysis, it is possible that the second metal-ion could be lost during
turnover. In fact, the kinetics of the hydrolysis of benzylpenicillin
catalysed by the cobalt substituted β-lactamase from B. cereus (BcII) are biphasic with an
initial burst of product formation followed by a steady-state rate of hydolysis
[75]. This was interpreted as being due to a branched kinetic pathway with two
enzyme intermediate species, ES1 and ES2, which have
different metal: enzyme stoichiometries. ES1 is a dicobalt enzyme
intermediate and is catalytic, but is slowly losing one bound cobalt ion during
turnover via the branching route, to give the mononuclear and inactive enzyme
intermediate ES2. The dependence of enzyme activity on pH and metal-ion
concentration indicates that only the dicobalt enzyme is catalytically active. The
monocobalt enzyme species, formed during the catalytic cycle, is virtually
inactive and requires the association of another cobalt ion for turnover. The
dicobalt enzyme intermediate is responsible for the direct catalytic route,
which is pH-independent between 5.5 and 9.5. The inactivation pathway of
metal-ion dissociation occurs by both an acid catalysed and a pH-independent reaction,
which is dependent on the presence of an enzyme residue of pKa 8.9
in its protonated form and shows a large kinetic solvent isotope effect (H2O/D2O)
of 5.2, indicative of a rate limiting proton transfer [75]. The pseudo
first-order rate constant to regenerate the dicobalt β-lactamase from
the monocobalt enzyme intermediate has a first-order dependence on cobalt ion
concentration. This unusual behaviour is attributed to an intrinsic property of
metallo hydrolytic enzymes that depend on a metal-bound water both as a ligand
for the second metal-ion and as the nucleophile which is consumed during
hydrolysis of the substrate and so has to be replaced to maintain the catalytic
cycle [75, 76].There remain questions regarding the necessity of the binuclear metal
centre itself: (i) are metallo-enzyme species responsible for the physiological
activity, the binuclear one or the mononuclear one, or are they interchangeable
under certain conditions? and if so, (ii) are these changes relevant to any
other functions, apart from catalysis, such as regulation of activity? It
does seem intrinsically odd in the binuclear enzyme that the bridging
hydroxide-ion is used as a nucleophile because of its presumed weak basicity.
Hydroxide-ion has three lone pairs, two of which are taken up by metal-ion
coordination and one of which is required for nucleophilic attack and covalent
bond formation. If this bridging hydroxide-ion is used as the attacking
nucleophile is it activated by general base catalysis so that concerted proton
removal generates a dianionic tetrahedral intermediate?



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*Source: 576297-2008-06-04.xml*