PROTEINS: Structure, Function, and Genetics 31:42–60 (1998)
Modeling of Inhibitor–Metalloenzyme Interactions and
Selectivity Using Molecular Mechanics Grounded in
Quantum Chemistry
David R. Garmer,1 Nohad Gresh,2* and Bernard-Pierre Roques2
1Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York
2USA Département de Pharmacochimie Moléculaire et Structurale, URA D1500 CNRS, U266 INSERM,
UFR des Sciences Pharmaceutiques et Biologiques, Paris, France
ABSTRACT We investigated the binding
properties of the metalloprotease inhibitors
hydroxamate, methanethiolate, and meth-
ylphosphoramidate to a model coordination
site occurring in several Zn21 metalloprote-
ases, including thermolysin. This was carried
out using both the SIBFA (sum of interactions
between fragments ab initio-computed) mo-
lecular mechanics and the SCF/MP2 proce-
dures for the purpose of evaluating SIBFA as a
metalloenzyme modeling tool. The energy-
minimized structures were closely similar to
the X-ray crystallographic structures of re-
lated thermolysin-inhibitor complexes. We
found that selectivity between alternative ge-
ometries and between inhibitors usually
stemmed from multiple interaction compo-
nents included in SIBFA. The binding strength
sequence is hydroxamate G methanethiolate
H methylphosphoramidate from multiple inter-
action components included in SIBFA. The
trends in interaction energy components, rank-
ings, and preferences for mono- or bidentate
binding were consistent in both computational
procedures. We also compared the Zn21 vs.
Mg21 selectivities in several other polycoordi-
nated sites having various ‘‘hard’’ and ‘‘soft’’
qualities. This included a hexahydrate, a model
representing Mg21/Ca21 binding sites, a chlo-
rophyll-like structure, and a zinc finger model.
The latter three favor Zn21 over Mg21 by a
greater degree than the hydrated state, but the
selectivity varies widely according to the li-
gand ‘‘softness.’’ SIBFA was able to match the
ab initio binding energies by F2%, with the
SIBFA terms representing dispersion and
charge-transfer contributing the most to Zn21/
Mg21 selectivity. These results showed this
procedure to be a very capable modeling tool
for metalloenzyme problems, in this case giv-
ing valuable information about details and
limitations of ‘‘hard’’ and ‘‘soft’’ selectivity
trends. Proteins 31:42–60, 1998.
r 1998 Wiley-Liss, Inc.
r 1998 WILEY-LISS, INC.
Key words: quantum chemistry; molecular
mechanics; inhibitor; metalloen-
zyme complexes; selectivity
INTRODUCTION
The knowledge of three-dimensional structures of
an increasingly large number of biologically impor-
tant metalloproteases will prompt efforts in the
design of inhibitors with optimized electrostatic and
steric complementarities with the critical enzyme
cavity.1 Inhibitor docking is generally governed by
the anchoring of its anionic moiety to the active site
metal ion. Most of the inhibitors which are useful in
zinc-metalloproteases belong to the following catego-
ries: carboxylate; thiolate; phosphate, phosphonate,
or phosphoramidate; and hydroxamate.2 Metallopep-
tidase inhibitors are capable of regulating the levels
of peptides or biologically active hormones and some
of these are presently used in therapeutics. Thus,
inhibitors of the angiotensin converting enzyme
(ACE) are endowed with antihypertensive activi-
ties.3,4 Neutral endopeptidase (NEP, EC 24.11) is
involved in the degradation of enkephalin in the
central nervous system and in inactivation of the
atrial natriuretic peptide,1 so that selective NEP
inhibitors are endowed with analgesic properties1,5
and with natriuretic and diuretic properties.1 One of
these inhibitors, thiorphan,5a is used as a new anti-
secretory agent. Current efforts are also directed
toward the design of inhibitors of collagenase to yield
molecules with antiarthritic properties,6 of inhibi-
tors of the matrix metalloproteases stromelysin and
astacin to oppose tumor cell invasion and metasta-
sis,7 of inhibitors of carbonic anhydrase to yield
drugs against glaucoma,8 etc.
In this study, we evaluated the quality of some
modeling methods which may be used to represent
the interactions of inhibitor molecules with metallo-
*Correspondence to: Nohad Gresh, USA Département de
Pharmacochimie Moléculaire et Structurale, URA D1500 CNRS,
U266 INSERM, UFR des Sciences Pharmaceutiques et Bi-
ologiques, 4, Avenue de l’Observatoire, 75270 Paris Cedex 06,
France. E-mail: gresh@bisance.citi2.fr
Received 3 March 1997; Accepted 10 October 1997
 MODELING OF INHIBITOR–METALLOENZYME INTERACTIONS
enzymes, and so provide tools for drug-design appli-
cations. We will principally focus on the SIBFA (sum
of interactions between fragments ab initio-com-
puted) molecular mechanics system, a relatively
inexpensive but accurate force-field model formu-
lated and calibrated to reproduce the results of ab
initio quantum chemistry.9 The benchmarks here
will be made with the SCF/MP2 supermolecule
procedure because this allows us to compare poten-
tial energy surfaces and distinct geometries not yet
found in crystallographic analyses.
SIBFA is based on a functional representation of
distinct electronic effects which control intramolecu-
lar and intermolecular interactions. The primary
intermolecular functions give numerical values for
electrostatic, exchange repulsion, dispersion, polar-
ization, and charge transfer interactions for a given
system geometry. The latter two are defined to have
a strong nonadditive component in many situations.
We have recently been able to obtain numerical
values for these terms directly from electronic wave
functions by using an analysis called the restricted
variational space method (RVS), developed by
Stevens and Fink.10 This provides an additional
check on the fundamental physical accuracy of the
molecular modeling methods discussed below. Re-
sults so far from calibrating the SIBFA model have
been highly encouraging,9e–g but have not included a
variety of realistic protein model systems that are
examined in this study.
The first trials will analyze the inhibitor binding to
a model Zn21 site similar to the active sites of
thermolysin and NEP. We will compare the binding
affinities of three distinct categories of inhibitors to
this model site, evaluate the selective contributions
of separate energy components, and see how well the
energy-minimized geometries compare to related
X-ray diffraction results.11–14 These trials will show
that the polarization and charge-transfer compo-
nents are strongly nonadditive here because of the
presence of several anionic ligands interacting with
the dication. This is anticipated to defeat the applica-
tion of conventional force fields which deny the
existence of most types of nonadditive quantum
electronic effects.
In the second part of this study, we will show an
application of modeling to examine the selectivity of
several polycoordinated complexes. We wish to evalu-
ate whether we can account for all of their discrimi-
natory properties in terms of specific energy compo-
nents and see whether the SIBFA model is successful
in making accurate predictions. For this purpose, we
will consider sites of widely different structure and
varying ‘‘softness’’ including a ‘‘zinc finger’’ binding
site15 and a chlorophyll-like structure. An examina-
tion of the origins of ion selectivity in terms of
interaction components will help to evaluate the
detailed usefulness of the hard-and-soft acid base
43
concept,16 which has been criticized in biochemical
contexts.17
The modeling results presented below show that
the SIBFA model reproduces the results of high-level
quantum chemical calculations carried out for model
coordination sites of several different types. The
coordination structures determined by energy optimi-
zations are also very similar to examples of protein
and small-molecule geometries. This includes ex-
amples of heme coordination sites in which SIBFA
also reproduces the form of difficult potential energy
surfaces. Therefore, the force-field model can be used
effectively for modeling problems in concert with
such techniques as energy minimization (used here)
plus simulated annealing and Monte Carlo.
COMPUTATIONAL PROCEDURE
The intermolecular interactions are computed with
the SIBFA procedure,9 which is formulated and
calibrated on the basis of ab initio SCF supermol-
ecule computations and extended to intramolecular
(conformational) energy computations.9a–b Refine-
ments to SIBFA were recently presented9f–g in light
of a series of results on divalent cation-ligand com-
plexes using an extended basis set,18 leading to an
improved representation of the second-order
terms.9f–g An important requisite is the ability of this
procedure to reproduce the individual components of
the SCF interaction energy, not just the total interac-
tion. This alone can ensure transferability of the
procedure and the prospect of accounting for nonad-
ditivity in complexes involving more than two mol-
ecules. In the results presented here, the internal
bond lengths and angles of functional groups were
frozen, but all of the intergroup and group-metal
structural parameters were optimized for minimum
energy. As in our earlier investigations,9e–h numeri-
cal results for the ab initio SCF energy components
of interactions were obtained using a procedure
developed by Stevens and Fink10 called reduced
variational space (RVS) analysis.
The RVS analysis has conventional definitions of
coulomb and exchange-repulsion interactions, which
is the same as the component analysis published in
1970 by Dreyfus and Pullman,19 Kollman and Allen,20
and Morokuma.21 A definition of polarization interac-
tion is obtained by having electron densities of
groups relax from the unperturbed states under the
influence of interacting groups. A definition of charge
transfer comes from the additional relaxation ob-
tained when occupied and virtual orbitals of interact-
ing species are allowed to mix. For example, the
dative bonding orbitals of a ligand significantly relax
into the outer vacant orbitals available from a metal
ion. The RVS analysis and other quantum chemical
calculations were primarily carried out by using a
modified version of the GAMESS computer code.22
The basis sets and effective core potentials used23
are the same as those used in our previous
44 D.R. GARMER ET AL.
publications.9e–g,18 They were shown to provide, for
both cation-ligand18 and H-bonded interactions,9c
binding energies in very good agreement with those
of larger-basis set calculations and experimental
values whenever available.
The enhanced double-zeta-plus polarization (DZP)
basis set is at the limit of what was affordable for the
complete coordination structures described below.
For example, the largest calculations on ion-heme
structures required between 400 and 440 basis func-
tions. Correlation energy estimates were obtained
with MP2 calculations24 and these were also cor-
rected for the basis set superposition error (BSSE).25
The definition of correlation energy is the differ-
ence of Hartree-Fock and MP2 interactions after
correction for BSSE. The effect of electron correla-
tion on intermolecular and metal–ligand interac-
tions appears to be dominated by dispersion-like
interactions, which increase interaction strengths
while having small effects in shortening contacts.
Defining these components from quantum chemistry
was necessary to validate the corresponding force-
field terms, but the components also provide insight
into the nature of some selectivity effects.
Computation of the Intermolecular
Interaction Energies
Within the SIBFA procedure, the intermolecular
interaction energy term DE is computed as a sum of
five separate contributions:
DE 5 EMTP 1 Erep 1 Epol 1 Ect 1 Edisp .
Although separate functions, these terms have some
interdependence, as described below and in Refer-
ence 9f. EMTP denotes the electrostatic (multipolar)
energy contribution, computed with distributed mul-
tipoles derived from the ab initio SCF wave function
of the constitutive fragments. The multipoles (up to
quadrupoles) are distributed on the atoms and bond
barycenters using the procedure developed by Vigné-
Maeder and Claverie.26 Erep is the short-range repul-
sion energy, computed as a sum of bond–bond, bond–
lone pair, and lone pair–lone pair interactions. Epol is
the polarization energy contribution, computed with
distributed, anisotropic polarizabilities on the indi-
vidual molecules. The polarizabilities are distrib-
uted on the centroids of the localized orbitals (het-
eroatom lone pairs and bond barycenters) using a
procedure of Garmer and Stevens.27 A Gaussian
screening of the polarizing field is used. The field
polarizing each molecule is computed with the perma-
nent multipoles and the induced dipoles of all the
other molecules in an iterative fashion. No induced
dipoles are used on the Zn21 cation, on account of its
very small polarizability, and consistent with the
fact that its own polarization energy in polyligated
complexes is negligibly small.18 Ect is the charge-
transfer energy contribution, in which a coupling
with the polarization is introduced.9f Edisp is the
dispersion energy contribution. Using the formula-
tion developed by Creuzet et al.,28 it is computed as a
sum of 1/R**6, 1/R**8 and 1/R**10 terms. These terms are
reduced at short distances by means of an exponen-
tial damping term. An explicit exchange-dispersion
term is introduced in the case of the mutual interac-
tions between polyatomic molecules. Directionality
effects are accounted for by the explicit introduction
of fictitious atoms with reduced van der Waals radii
to represent the lone pairs. As in our previous
studies,9e–g the ab initio SCF computations on the
individual molecules, necessary to derive the distrib-
uted multipoles and polarizabilities, were done with
the Stevens et al.23 basis set, namely, the same as
used in the intermolecular ab initio ones.
Computation of the Continuum
Solvation Energies
The solvation energies are computed using the
Langlet et al. continuum procedure.29 Within this
procedure, the solvation energy is computed as the
following sum:
Esolv 5 Ecav 1 Eel 1 Epol 1 Edr .
Ecav is the cavitation energy, calculated as a sum of
contributions from intersecting atoms, centered on
the solute atoms, using a Reiss-Pierotti formula. Eel
is the solute-solvent electrostatic energy. The electro-
static potential generated by the solute is computed
with the help of the same distributed multipoles as
in the expression of EMTP. The dipoles, which are
induced on the polarizable centers of the solute
molecules, are taken into account in the computation
of Eel along with the permanent mutipoles. At this
stage, however, the additional dipoles induced on the
solute molecules because of the reaction field of the
solvent are not taken into account. Such a refine-
ment will be introduced in later treatments (Langlet,
J., work in progress). Epol is the solute polarization
energy and Edr is the dispersion-repulsion contribu-
tion. As discussed in Reference 30, this procedure
enables one to compute both the ‘‘free energy’’ term,
denoted as DGhydr below, and its enthalpic contribu-
tion, denoted as DHhydr below.
Calibration
The SIBFA parameters are derived on a functional
group basis with most of the parameters required for
this work being previously available. The novel
groups, which were introduced in the present study,
are the porphinato anion, the phosphorus-contain-
ing inhibitors represented in Figure 1, and methylhy-
droxamate.
The highly polarizable nature of the porphinato
anion, having two negative charges delocalized over
the four imidazolates and their connecting etheno
bridges, required a different screening than imidaz-
 MODELING OF INHIBITOR–METALLOENZYME INTERACTIONS
Fig. 1. Molecular structures of methylphosphoramidate, dimethylphosphonate, and dimethylphos-
phinate.
ole. This was done by adopting values of the F
parameter, the Gaussian exponent of the screening
functional,9f of 0.85 and 0.50, respectively, for the
bond and lone pair polarizabilities instead of the
corresponding values of 1.40 and 1.95 in imidazole.9f
The values were selected in order to reproduce the ab
initio SCF value of DE for the interaction of a single
Zn21 cation in the middle of the porphinato ring.
The functional representations of Erep, Epol, Ect,
and Edisp in SIBFA for the phosphorus-containing
inhibitors required effective radii definitions for the
anionic oxygen atoms. As in Reference 9g, these were
developed by fitting to the corresponding RVS and
Moller-Plesset (MP2) terms for a Zn21–H2PO4-
bidentate complex at a few different coordination
distances. The values of effective radii for Erep, Epol,
Ect, and Edisp are 1.65, 1.625, 1.85, and 1.65 A,
respectively, a set of radii comparable to the corre-
sponding one of 1.50, 1.625, 1.85, and 1.50 A for
carboxylate oxygens.9f The K(Zn-P) coefficient of Edisp
was fit in order to reproduce the values of the
correlation energy, Ecorr, from MP2 computations on
the Zn21–H2PO4- bidentate complex in the range of
explored coordination distances.
No recalibration was done for our SIBFA calcula-
tions on the Zn21 methylhydroxamate complexes.
The C-bound anionic O was given the same effective
radii as the formate O’s, and the N-bound anionic O
was given the same radii as the methoxy O’s. The
other ligands, water, formaldehyde, and methanethi-
olate, were taken from our SIBFA library of frag-
ments determined with the Stevens et al. basis set.
Energy-minimization using SIBFA resorted to the
polyvalent minimizer ‘‘Merlin.’’31
The example of phosphorus-containing ligands
illustrates that the calibration which has been done
on one representative member of the class, namely,
H2PO4-, is transferable to the other ones, namely,
methylphosphoramidate, dimethylphosphonate, and
dimethylphosphinate. The only requisite is to derive
their ab initio multipoles and polarizabilities. This
has to be done only once for each novel ligand, which
can then be integrated to the SIBFA library of
45
fragments, and also used as a constitutive building-
block within larger molecular entities.
RESULTS
Results From Inhibitor Structures
This section first presents tests of the interactions
of inhibitors with the zinc dication to investigate the
behavior of the SIBFA model. In this we compare the
individual components and total binding energy
estimates for several geometric modes. Subse-
quently, calculations of several structures represent-
ing the first shell of the enzymes thermolysin and
NEP will be discussed. These test the behavior of the
force field in macromolecular assemblies and can
easily be extended to investigate interactions with
the rest of the enzyme. Methylphosphoramidate,
dimethylphosphonate, dimethylphosphinate, metha-
nethiolate, and methylhydroxamate are representa-
tives of important classes of inhibitors used primar-
ily for zinc enzymes.
Phosphorus-containing inhibitors
In order to verify the transferability of the results,
we report in Table I the binding energies of meth-
ylphosphoramidate, dimethylphosphonate, and di-
methylphosphinate to Zn21 in optimized geom-
etries. In this and the following tables we denote by
DEO/SIBFA the SIBFA values without the Edisp
component. This term is to be compared to the ab
initio DE/SCF term, before inclusion of Ecorr.
The SIBFA computations are seen to match the ab
initio ones to within 2%, although the energetics for
replacement reactions are less accurate. The major
errors are for estimates of Epol and Ecorr for particular
ligands.
Hydroxamate
The hydroxamate anion is the anionic moiety of
several pharmacologically important molecules,
which can be linked to its very strong affinity for
Zn21. Thus, hydroxamate-containing compounds
which are inhibitors of matrix Zn21 metalloprote-
46 D.R. GARMER ET AL.

TABLE I. Ab Initio vs. SIBFA Binding Energies (DE) TABLE II. Compared Ab Initio and SIBFA Binding
of P-O Functional Groups to Zn21 Energies Energies of Zn21 With Methylhydroxamate;
in kcal/mol Energies in kcal/mol

Methyl- Dimethyl- Dimethyl- Ab initio SIBFA

Ab initio and phosphora- phospho- phosphi- E1 2312.3 2312.8
RVS terms midate nate nate Epol 295.1 284.1
E1 2283.6 2267.5 2281.6 Ect 223.5 232.4
Epol 293.1 291.1 293.1 DE/SCF 2430.9
Ect 223.0 222.0 223.1 DE0/SIBFA 2429.3
DE/SCF 2399.7 2380.6 2397.8 Ecorr 28.1
Ecorr 217.0 216.9 215.5 Edisp 221.8
DE/MP2 2416.7 2397.5 2413.3 Total DE 2439.0 2451.2

SIBFA terms
E1 2282.9 2265.8 2280.9
Epol 282.0 281.6 284.3
Ect 227.3 226.8 223.8
DE0/SIBFA 2392.2 2374.3 2389.0
Edisp 220.0 220.8 219.7
DE/SIBFA 2413.1 2395.1 2408.7
ases can behave as antitumor agents.7 No specific
recalibration was done for our SIBFA calculations on
the Zn21 methylhydroxamate complexes except for
calculation of the methylhydroxamate electrostatic
multipoles and polarizabilities. Effective radii were
taken from other related species computed earlier.9e–g
Energy-minimization using SIBFA resulted in a
deep minimum, in the O-C-N-O plane, in a bidentate
position with respect to the two anionic O’s (d Zn-O 5
1.83 Å). A single-point ab initio computation was
then performed on this position and the compared
SIBFA vs. ab initio energy results are given in
Table II.
Both ab initio and SIBFA computations show
methylhydroxamate to be endowed with consider-
ably stronger binding affinities for Zn21 than meth-
ylphosphoramidate and dimethylphosphinate, but
there is some variation in the reproduction quality of
individual components. The ab initio energy compo-
nents for a replacement of methylphosphoramidate
with methylhydroxamate are 228.7 from E1, 22.6
from Epol, 20.5 from Ect, and 8.9 kcal/mol from Ecorr.
The corresponding values in SIBFA are 229.9, 22.1,
25.1, and 21.8 kcal/mol. The Zn21 methylhydroxa-
mate affinities are also stronger than those of metha-
nethiolate, which amounts to 2424 kcal/mol in both
MP218a and SIBFA9g computations.
We performed a series of in- and out-of-plane
variations of the position of Zn21 with respect to
methylhydroxamate, at a fixed distance from one
anionic oxygen, and compared the SIBFA computa-
tions (without Edisp) to the SCF ones. A representa-
tive curve is given in Figure 2, in which we plotted
the evolutions of DE/SCF and DE/SIBFA and their
E1, Epol, and Ect components as a function of out-of-
plane variations of Zn21. Zn21 rotates on a cone
defined by d(O–Zn) 5 1.8 Å, and u (N-O-Zn) 5 120°.
The loss of binding energy on departing from biden-
tate Zn21 binding is much larger than in the Zn21
carboxyl or phosphate complexes. We also note that
the SIBFA binding energy curve and components can
closely reproduce the SCF ones in the energetically
relevant zone.
Inhibitors and thermolysin/NEP coordination
These protein active sites have a zinc ion coordi-
nate with two histidine sidechains and glutamate
with open sites for catalytic activity.11 We will denote
by Zn-HHEX, Zn-HHES, and Zn-HHEP the com-
plexes of Zn21 in its TLN/NEP-like binding site with
methylhydroxamate (X), methanethiolate (S), or
methylphosphoramidate (P), respectively.
The H, E, and X ligands were first set with the help
of a computer graphics program in the same posi-
tions around Zn21 as in the X-ray structure of TLN
bound to a hydroxamate inhibitor.14 This was used as
a starting point for energy minimizations. In the
SIBFA energy-minimized structure of Figure 3a, and
similar to the X-ray structure, Zn21 is bound in a
monodentate fashion to the carboxyl, whereas it is
bidentate-bound to methylhydroxamate. A compet-
ing structure was generated by imposing distance
restraints between Zn21 and the two formate an-
ionic oxygens to enforce a bidentate coordination.
The converged structure after energy minimization
is given in Figure 3b. Energetics favor the five-
coordinate structure shown in Table III, and releas-
ing the constraint causes the second to rearrange to
match the first. This is true for the force-field model
and ab initio calculations, as an energy minimization
at the SCF level gave a geometry practically identi-
cal to the SIBFA result.
Table III shows the SIBFA results very closely
reproduce the ab initio ones:
● The monodentate binding mode to formate is
favored over the bidentate one by very similar
amounts (4–6 kcal/mol out of 700) in both proce-
dures.
● The numerical agreement between SIBFA and the
ab initio computations is within ,2%.
● In both procedures, the principal term responsible
for the mono- over bidentate preference is Epol (4-5
 MODELING OF INHIBITOR–METALLOENZYME INTERACTIONS 47

Fig. 2. Zn21 methylhydroxamate complex. Compared evolutions of the ab initio SCF and SIBFA
energies and their components as a function of out-of-plane variations of Zn21.

kcal/mol), whereas E1 contributes to it by a mod-
est amount (1 kcal/mol), and Ecorr/Edisp bring an
opposite, but weak (,0.8 kcal/mol) contribution.
● Again in both procedures, both Ect and Epol have
decreased in absolute values with respect to the
monoligated Zn21–methylhydroxamate (compare
with Table II). This is particularly striking in the
case of Ect, which has undergone a decrease by a
factor of 2, whereas Epol has correspondingly de-
creased by a factor of 15%.
This is to be contrasted with the case of the
complexes of Zn21 with a first-shell of neutral
ligands, a typical example of which is Zn21
(H20)4.9f,18b In that case, both Epol and Ect continued
to undergo increases in their magnitudes with re-
spect to the monoligated Zn21 (H20) complex at an
equivalent O-Zn distance. Nevertheless, the ampli-
tude of this increase was limited to factors of 2.8 and
2.0, whereas the number of ligands was passing from
one to four, indicative of a saturation tendency of the
cation’s virtual orbitals. In the present case, it
appears that inclusion of negatively-charged ligands
in the cation first-shell will produce an actual inhibi-
tion of the cation’s electron-acceptor character.
These results and similar trends for other ex-
amples demonstrate a much greater sensitivity of Ect
than Epol to saturation of the cation’s ligation shell.
Fundamental differences in behavior such as this
account for some of the difficulty in using simple
force-field models to describe ionic systems.
We wished to quantify the energetical contribution
of methylhydroxamate to the stabilization of the
Zn21 binding site in the presence of solvation ef-
fects. For that purpose, we progressively moved
methylhydroxamate away from the Zn21 binding
site and we monitored the evolutions of DE(SIBFA),
DHhydr, DGhydr, DE 1 DHhydr, and DE 1 DGhydr as a
function of the d(Zn-O) distance, the cation remain-
ing equidistant to its two anionic oxygens. These
evolutions are plotted in Figure 4. Both DE 1 DHhydr
and DE 1 DGhydr have a smooth decrease (in absolute
values) upon increasing d, due to the mutual compen-
sations between the steep decrease of DE, on the one
hand, and concomitant increases of DHhydr and DGhydr
on the other hand. These features are similar to the
corresponding ones found upon separating away
formate and methylammonium from their zwitteri-
onic complex to infinite separation, previously inves-
tigated by us.30 In the present case, the optimized
48 D.R. GARMER ET AL.

Fig. 3. Energy-minimized structures of the complexes of the anionic moieties of inhibitors to

Zn21 in a binding site made out of two imidazoles and one formate. (a) Zn-HHEX complex, Zn21
monodentate to formate; (b) Zn-HHEX complex, Zn21 bidentate to formate; (c) Zn-HHES complex,
Zn21 monodentate to formate; (d) Zn-HHES complex, Zn21 bidentate to formate; (e) Zn-HHEP
complex, Zn21 monodentate to formate; (f) Zn-HHEP complex, Zn21 bidentate to formate.
 MODELING OF INHIBITOR–METALLOENZYME INTERACTIONS 49

TABLE III. Values of the SIBFA and Ab Initio Both also relax to these geometries with monoden-
Binding Energies in Five- and Six-Coordinate tate formate if unrestrained energy minimization is
Zn-HHEX Model Geometries carried out. This was not observed in AMBER compu-
5-Coordinate 6-Coordinate tations bearing on the complexes of TLN with phos-
phoramidate inhibitors, in which unrestrained mo-
Ab initio
lecular dynamics converged to sixfold coordinated
E1 2552.1 2550.7 kcal/mol
Epol 283.1 279.0 arrangements around Zn21 with a bidentate-bound
Ect 212.2 212.9 carboxylate.32 The SIBFA-generated geometries are
DE/SCF 2651.6 2647.0 shown in Figures 3c–f.
We have collected in Table IV the SIBFA energy
Ecorr 254.1 254.9 results for these complexes, including estimates of
DE/MP2 2705.7 2701.9 DH and DG of hydration from the Langlet-Claverie
continuum method. The agreement with the corre-
SIBFA sponding ab initio results is here also very good,
EMTP 2663.5 2658.5
except for the estimates of electron correlation ef-
Erep 107.8 104.3
E1 2555.7 2554.2 fects by Edisp, which may need some improvement.
Epol 273.4 268.8 Both calculations show that the preference for a
Ect 217.0 217.2 lower coordination number is smallest for Zn-HHES,
DE0/SIBFA 2646.1 2640.2 for example, but the stability order for the Zn-HHES
and HHEP structures differ after electron correla-
Edisp 250.5 250.9 tion is taken into account.
DE/SIBFA 2696.6 2691.0 Tables III and IV also show that the hydration
terms invariably favor arrangements of lower coordi-
DGhydr 255.4 250.6
nation numbers, due to the greater exposure of a
DHhydr 285.5 279.9
formate oxygen to solvation. In enzymatic active
sites, however, the contribution of DG/DHhydr to the
energy balances will depend on the structure of its
Zn21 methylhydroxamate distance is increased from actual inhibitor’s complex, and the modulation of its
2.0 A for DE to 2.8 A for both DE 1 DHhydr and DE 1 remaining accessibility to water. This was recently
DGhydr. For such an equilibrium distance, DE 1 observed by us in simulations of the complexes of
DHhydr and DE 1 DGhydr are, respectively, 33.5 and thiorphan and of a phosphoramidate inhibitor to the
35.7 kcal/mol stronger than at infinite separation of active site of a native vs. a mutated cavity of TLN.33
methylhydroxamate. This implies that the preferred However, the use of a continuum model can still
geometry in an aqueous environment lacks direct greatly speed up the process of the evaluation of
bonding contacts of the methylhydroxamate ion with different structures.
its binding site. This prediction is probably not The stability ordering of the three arrangements
completely accurate, since chemically related com- from MP2 results is:
plexes with direct bonding have been prepared by
inorganic chemists many times. Therefore, further Zn-HHEX . Zn-HHEP . Zn-HHES.
improvements and testing should be made on this 10 2 kcal/mol (1)
point, possibly by including a limited number of
explicit water molecules in trial calculations. Whereas, with respect to the monoligated com-
The Zn-HHES and Zn-HHEP complexes were gen- plexes, methylhydroxamate remains the ligand giv-
erated by substituting methylhydroxamate with ing rise to the most stable complex, a reversal in the
methanethiolate and methylphosphoramidate, re- relative ordering of methylphosphoramidate vs.
spectively, which were positioned in the same orien- methanethiolate has occurred, which now favors the
tation as observed in the X-ray crystal structures of former. The ordering is not modified by taking into
the complexes of TLN with thiorphan,12b and phos- account solvation effects, for which an estimate is
phoramidon.13 Similar to Zn-HHEX, energy minimi- provided by the continuum procedure. Note that the
zation using the SIBFA procedure led to structures ordering at the SCF level favors by 4 kcal/mol out of
in which Zn21 remained monodentate-bound to 620 methanethiolate over phosphoramidate. The
formate. In Zn-HHEP, it is bidentate-bound to the values of DE0(SIBFA) in the absence of dispersion
two anionic oxygens of methylphosphoramidate. rank HHEX . HHEP . HHES with only a small (3.2
Competing structures with Zn21 bidentate bound kcal/mol out of 620) energy difference between HHEP
to formate were enforced by imposing a distance and HHES. DE(SIBFA) favors HHES over HHEP
restraint of 2.05 A on the two Zn-O(formate) dis- when Edisp is taken into account, but, again, by a
tances. After convergence and removal of the re- small margin (2.7 kcal/mol out of 680). This is
straint energy term, both structures were found to be because Edisp has a smaller value in HHEP than in
less stable than the completely optimized forms. HHES, whereas the converse occurs with Ecorr. This
50 D.R. GARMER ET AL.
Fig. 4. The Zn21 HHEX complex. Evolutions of DE, DHhydr, DGhydr, DE 1 DHhydr, and DE 1
DGhydr as a function of increasing the Zn21 methylhydroxamate distance.
translates again the need to further refine the formu-
lation of the SIBFA Edisp term, possibly by an analy-
sis of the extent of nonadditivity in polycoordinated
cation complexes.
Thus, methylhydroxamate is predicted to be the
most effective functional group to interact with the
first shell of this kind of zinc metalloenzyme. This
likely accounts for its ability to help inhibitors
disrupt zinc finger coordination.
However, this energetics does not directly include
the additional interactions occurring within the bind-
ing site. Thus, the phosphoramidate group of phos-
phoramidon was seen to partake in an H-bonding
interaction with the hydroxylic group of the proto-
nated Glul43 residue of TLN,13 whereas the metha-
nethiolate group of thiorphan did not.12 In this
connection, our recent simulation of the complexes of
thiorphan and a phosphoramidate inhibitor within
the active site of TLN33 using these methods gave a
more favorable binding energy DE for phosphorami-
date than thiorphan than could be anticipated from
the energies computed for methanethiolate with the
first shell. We will elaborate on this issue below.
Modeling of Other Sites and Metal Selectivity
This section is devoted to showing a cross-section
of results from the techniques discussed above ap-
plied to other prototypical ion binding sites. Quan-
tum chemistry and the RVS analysis method provide
energetics which should be comparable to experimen-
tal DG quantities, though with some inherent inaccu-
racy, but it also shows the physical origins of selectiv-
ity effects. This is important for an understanding of
real systems in which only some thermodynamic
quantities are measurable. The RVS analysis pro-
vides a good empirical basis for the correct applica-
tion of a qualitative rule such as HSAB to metal ion
binding. The application of the SIBFA molecular
modeling technique provides another rather strin-
gent test of the capabilities of the force field for
general molecular modeling problems.
We have so far applied these theoretical tech-
niques to five model systems, aside from the inhibi-
tor models discussed above (see Fig. 5). Although we
accumulated incomplete results for several other
cations using quantum chemistry and SIBFA, it
appears that the important conclusions can be estab-
lished by showing a complete series for the binding of
zinc and magnesium dications.
The structures included a model of hydration for
these dications consisting of an octahedral arrange-
ment of six coordinated water molecules. The next
two model structures were set up to reflect experi-
mentally observed magnesium binding sites of pro-
 MODELING OF INHIBITOR–METALLOENZYME INTERACTIONS 51

TABLE IV. Values of the Binding Energies in the Complexes of Zn21 With Two Imidazoles, One Formate, and
One Cysteinate (Zn-HHES), or One Methylphosphoramidate (Zn-HHEP) Ligand; Differences From Other
Inhibitor Complexes Are Also Shown

Zn-HHES Zn-HHEP
4-Coordinate 5-Coordinate (2HHEX) 5-Coordinate 6-Coordinate (2HHES)
Ab initio
DE/SCF 2627.7 2624.6 23.9 2623.6 2620.1 4.1

Ecorr 265.9 265.4 211.8 272.0 270.9 26.1

DE/MP2 2693.6 2690.0 12.1 2695.6 2691.0 22.0

SIBFA
EMTP 2636.0 2632.3 27.5 2643.2 2636.2 7.2
Erep 123.4 114.4 15.6 113.4 99.2 210.0
E1 2512.6 2517.9 43.1 2529.8 2537.1 217.2
Epol 288.4 281.0 215.0 280.6 272.3 7.8
Ect 222.6 222.7 25.6 216.5 216.0 6.1

DE0/SIBFA 2623.7 2621.6 22.4 2626.9 2625.4 23.2

Edisp 259.4 258.7 28.9 253.5 252.5 5.9

DE/SIBFA 2683.1 2680.3 13.5 2680.4 2678.0 2.7

DGhydr 247.8 244.3 7.6 252.0 251.8 24.2

DHhydr 275.6 271.5 9.9 283.0 282.7 27.4
Hydration energies for groups
H E X P S
DGhydr 27.5 288.0 293.6 287.0 282.0
DHhydr 216.1 298.0 2107.6 298.0 292.0

teins or some inorganic crystals. These structures
have two water molecules and two formaldehyde
units, representing carbonyl coordination, coordi-
nated as in proteins. The shells are completed by
either two formate or two fluoride anions. These
structures were verified to be local minimum struc-
tures for the SCF-level quantum chemistry and the
SIBFA force field. The structural details are shown
in Figure 5 for Zn21 coordination, with the SIBFA-
optimized estimates for some internal coordinates in
parenthesis.
Another model system had coordination of two
imidazole ligands and two methanethiolate anions.
The detailed optimized structures are close mimics
of the coordination observed for Zn21 in zinc finger
proteins. Thiolate functional groups have never been
observed to make coordination bonds to Mg21 but
the SCF optimization still does indicate a local
energy minimum with very long coordination bonds
of 2.40 Å for Mg-S.
The last sort of system we considered is a model
heme site. These structures were set up to have an
approximately symmetrical porphinate core with an
axial water ligand. The SCF and SIBFA optimized
structures with magnesium or zinc are very similar
in having a small, approximately 0. 15 A displace-
ment of the central ion from the mean atomic plane
of the heme unit. This is in accord with structural
details for similar pentacoordinate heme systems
known from crystallography (see Refs. 34 and 35 for
a useful analysis of cation–heme structures). In the
ab initio computations, the porphinato ring systems
are also slightly domed upward, giving the appear-
ance of a meniscus around the displaced cations.35
The binding energy of the axial water ligands were
determined by also calculating the cation–porphi-
nate optimal geometries. These were fully planar,
although the optimizations were started off from the
domed geometries.
Table V shows the total binding energies for this
set of structures with respect to completely sepa-
rated groups. Figure 6a,b give the estimated thermo-
dynamic results of selectivity reactions of the form of
Mg21X = Zn21X in which X is a model binding site.
The thermodynamic components of the selectivity
equation are also given as computed by the RVS
analysis or from the SIBFA terms. The SIBFA en-
tries are incomplete because the required fluoride
ion parameters have not yet been developed. How-
ever, we have included estimates of the external
solvation thermodynamics for the SIBFA calcula-
tions.
Table V shows that the SIBFA computations are
able to reproduce the ab initio results in the com-
plexes of Zn21 and Mg21 with A, B, and D. This is
very encouraging, considering the various strong
52 D.R. GARMER ET AL.

Fig. 5. Structures B-E which were used to examine Zn/Mg selectivity origins. Some Hartree-Fock-
optimized distances are shown with the corresponding SIBFA results in parentheses.

local electrostatic effects which come into play and
the high ligand polarizabilities, giving rise to strong
nonadditive effects. At the SCF level, the match is to
within ,3%. It is not downgraded at the MP2 level,
partly because some offset at the SCF level is recov-
ered to some extent at the MP2 one. The least
agreement between the SIBFA and the ab initio
results exists for the water-porphinato anion struc-
tures.
The hexahydrate binding site model, A, is the least
selective between Mg21 and Zn21 in total binding
energy, the difference favoring Zn21 being 21.6 and
30.3 kcal/mol in the MP2 and SIBFA computations,
respectively. These values are close to the experimen-
 MODELING OF INHIBITOR–METALLOENZYME INTERACTIONS 53

TABLE V. Energy Components for Several Model Binding Sites Computed From the SIBFA Force Field and
MP2 Quantum Chemistry With a BSSE Correction

Mg2 1 A Zn2 1 A Mg2 1 B Zn2 1 B Mg2 1 C Zn2 1 C Mg2 1 D Zn2 1 D*

Ab initio
DE/SCF 2316.0 2319.6 2639.1 2645.8 2709.8 2719.6 2602.0 2627.4

Ecorr 29.0 227.0 226.8 248.7 23.5 225.7 215.8 265.8

DE/MP2 2325.0 2346.6 2665.9 2694.5 2713.3 2745.3 2617.8 2693.2

SIBFA
EMTP 2288.6 2295.2 2672.5 2678.9 2581.6 2585.6 2600.0 2629.0
Erep 59.1 77.4 110.4 132.3 56.5 58.9 95.6 131.7
E1 2229.6 2217.9 2562.1 2546.6 2525.1 2526.6 2504.4 2497.2
Epol 275.1 279.1 267.9 272.1 2161.8 2161.8 287.7 292.4
Ect 24.3 214.6 212.3 222.3 24.0 211.8 25.1 226.2

DE0/SIBFA 2308.3 2311.6 2642.3 2641.0 2690.9 2700.2 2597.2 2615.8

Edisp 28.5 236.2 220.7 257.4 24.3 242.8 210.2 269.1

DE/SIBFA 2317.5 2347.8 2663.0 2698.4 2695.2 2743.1 2607.4 2684.9

DGhydr 2208.7 2207.9 242.3 242.3 2176.4 2173.1 247.9 243.2

DHhydr 2232.0 2230.7 265.7 265.5 2235.8 2232.4 277.7 271.3
*Complex A is a hexahydrate, B is a model Mg/Ca binding site with (water)2(formaldehyde) 2 (formate)2 shell, C is porphinate with
axial water, and D is a zinc finger model with (methanethiolate)2 (imidazole)2 .

tal difference of 30 kcal/mol in the dehydration
enthalpies of Zn21 vs. Mg21. This type of geometry
is assumed to model the hydrated state for these
cations. Therefore, the other protein-related binding
site models are predicted to produce an inherent
selectivity for Zn21 over Mg21.
This is true even for the model structure labeled B,
which resembles the coordination of some magne-
sium and calcium binding sites in proteins. Model B
is composed entirely from donors considered hard in
HSAB scales16 but the ab initio and SIBFA calcula-
tions show it to be 7 and 8 kcal/mol, respectively,
more selective for Zn21 than the hydration model A.
Since the structural details for Zn21 and Mg21 are
practically identical, the occupation of sites resem-
bling this one by hard cations in proteins is most
likely due to the much greater concentrations of
Mg21 and Ca21 in tissues compared to soft cations
such as Zn21.
In complex D, which is representative of cation
binding sites in zinc fingers, selectivity for Zn21 is
75.4 and 77.5 kcal/mol in the MP2 and SIBFA
computations, respectively. In contrast with sites A
and B, this selectivity is so great that Mg21 coordi-
nation is impossible under physiological conditions.
In terms of components, E1 remains favorable for
Mg21 over Zn21, Epol gives very little selectivity for
zinc, while Ect and Ecorr are very strongly in favor of
Zn21, both providing energy differences at least
doubled over sites A and B.
The magnitude of charge transfer energies is,
however, relatively small in these complexes, com-
pared to examples with only one ligand, because
polar and charged ligands inhibit charge transfer to
the dication from other ligands. Thus, a model like
SIBFA, having nonadditive charge transfer and po-
larization energetics, is required to address prob-
lems involving several types of available donors
competing for binding. The correlation effect, how-
ever, remains with large magnitudes. Ecorr is mod-
eled as a dispersion interaction in SIBFA, and en-
ables one to reproduce these energetics satisfactorily.
Selectivity of the heme-plus-axial water model for
Zn21 over Mg21 is only moderately greater than for
the hexahydrate. Thus, related magnesium–porphi-
nate structures such as in chlorophylls can be stable
against ion exchange in excess Mg21. The reproduc-
tion of quantum chemistry-predicted structural and
energetic properties by the SIBFA force field is
relatively poorer for this system, but still at least
qualitatively accurate. The force field also predicts
that the axial water ligand has a moderately strong
attraction for the central dication in agreement with
the Hartree-Fock and MP2 calculations (Table V).
Prediction of the binding properties of systems
such as these provides a very difficult test of theoreti-
cal methods because interaction energies, electrostat-
ics, and electronic polarization effects are very large.
In particular, the heme ring system electronic charge
distribution is heavily modified by binding a dica-
tion. However, various semi-empirical quantum
chemistry methods might provide another alterna-
tive for modeling studies. Therefore, we tested the
popular PM3, AM1, and MNDO methods imple-
mented in MOPAC versions 5 and 636 against the
54 D.R. GARMER ET AL.

Fig. 6. Ab initio (a) and SIBFA (b) binding energy trends in complexes ‘‘A,’’ ‘‘B,’’ ‘‘C,’’ and ‘‘D,’’
following the sequence of increasing softness.
 MODELING OF INHIBITOR–METALLOENZYME INTERACTIONS
Fig. 7. The Zn21–porphinato–water complex. Contribution of
the water molecule to the total stabilization energy as a function of
the Zn–O distance, as computed by the MP2, the SIBFA, and the
PM3 procedures.
MP2 results we have from a moderately large basis
set.
What we found from this comparison was that the
semi-empirical methods can provide qualitatively
incorrect structures for the potential energy surfaces
(PES). The example easiest to visualize was in the
PES for stretching the coordination of the axial
water molecule away from the center of the heme
system. This may be a significant PES for ligand
exchange processes necessary for enzyme assembly
and catalysis, so that we would like to have depend-
able theoretical models less expensive than ab initio
or density functional quantum chemistry. The water–
Zn21porphinate stretching PES from the various
theoretical methods are summarized in Figure 7.
The MP2 energetics calculated with a BSSE correc-
tion indicated a monotonic attraction for the axial
water, leading to a single energy minimum with an
appropriate coordination bond length. Ab initio Har-
tree-Fock calculations give a very similar PES with a
maximum binding energy of 16.5 kcal/mol. The PES
structure was essentially duplicated by the SIBFA
force field, although the bond strength is somewhat
weaker than the MP2 result.
In contrast, the semi-empirical methods all pre-
dicted that a PES barrier exists, separating the
coordination structure from a secondary regime with
weak interaction of the axial water. Outside of the
PES barrier feature, the water molecule was rolled
to lie almost parallel to the ring system but the
Zn–oxygen interaction was still the closest contact.
Characteristics of the PM3 PES are shown but the
AM1 and MNDO PES’s have a similar structure,
with the barrier at about 3 A. All three models give
positive interaction energies for the axial water at all
distances, including in the coordination bond struc-
55
ture. We tested similar PES surfaces for several
other axial ligands and cations, and for some ex-
amples with two axial ligands. Most of these had
similar barrier features and some even had second-
ary energy minima at very long coordination dis-
tances.
All of this behavior is probably artificial, the result
of poor parametrization of nuclear interaction terms
in these semi-empirical methods. This can cause
problems in interpreting experimental data since
there are a few examples of heme-containing crystal
structures with very long coordination bonds for an
axial ligand.34 Semi-empirical calculations may
falsely suggest that this has an origin in electronic
properties of the cation–ligand interaction. This is
not the case with the SIBFA calculations, which
appear as a dependable alternative to the ab initio
procedure in such PES studies.
The last system we examined is shown as model E
in Figures 5 and 6a. Here the ionic component of the
first shell is two fluoride ions. This showed a moder-
ate but qualitative change in selectivity in the ab
initio results compared with related model B. The
fluoride-containing model shell is now comparable to
the hexahydrate in relative attraction for zinc vs.
magnesium. Fluoride has been considered a hard
donor species and these results suggest that it can
have a significant effect just as part of a coordination
shell. We also plan to investigate other species such
as R3PF- groups as donors to see if these have a
similar effect, since none of the other structures we
investigated appear to inherently have the ability to
competitively attract hard ions. Thus, covalently
bonded fluoride ions might be useful in controlling
the selectivity of ionophore compounds. This has not
been investigated experimentally, to our knowledge.
Stability Calculation Using
the Continuum Method
This section reports two types of calculations
which employ the continuum method combined with
the SIBFA force field calculations. The first are
stability estimates made for some of the model
structures and the second are estimates of the
inhibitor thermodynamics made for the binding site
of the protein thermolysin. We would consider these
calculations an alternative to the free energy pertur-
bation methods (reviewed in Ref. 37) which require
immense computational expense using force fields,
which are not as well grounded in physical theory as
SIBFA.
The stability of the complexes discussed above
may be obtained by subtracting the desolvation
energies of the ligands and cation from the DE 1
DHhydr estimate for a complex. The desolvation ener-
gies of the cations are estimated from the hexahy-
drates DE 1 DHhydr 2 6*DHhydr (water) for consis-
tency. These values are 2490.8 kcal/mol for Mg21
56 D.R. GARMER ET AL.
and 2519.8 kcal/mol for Zn21. The desolvation
enthalpies, DH, of the ligands are given in Table IV.
Those of water, formaldehyde, and the porphinato
anion are 29.7, 27.8, and 2277.7 kcal/mol, respec-
tively. The stability estimates (DE in solution) are
given below:
Zn-HHEX. Zn-D. Zn-HHES. Zn-HHEP. Zn-B
DE 1 DHhydr 2782.1 2756.2 2758.7 2763.4 2763.9
DHhydr(lig) 2237.8 2216.2 2222.2 2228.2 2231.0
DEsolution 224.5 220.2 216.7 215.4 213.1
Mg-B . Mg-D
DE 1 DHhydr 2728.7 2685.1
DGhydr(lig) 2231.0 2216.2
DEsolution 26.9 121.9
These results are well in line with our expectations. For
example, the Zn-inhibitor complexes and zinc finger
model D are very stable. The B site is less so, which is
expected since similar protein sites often allow for rapid
ion exchange. The never-observed Mg-zinc finger combi-
nation is predicted to have positive enthalpy of assem-
bly.
In the case of the cation–porphinato complexes,
however, the estimated DHhydr values were over 150
kcal/mol larger than for any of the other complexes.
This is due to the large solvent exposure of the
relatively open axial site and indicates the limits of
the validity of a continuum hydration estimate.
We then wished to evaluate the effect an actual
protein would exert on the energy ordering of the
three anionic moieties, as compared to the model
oligoligates considered above. For that purpose, we
docked them in the binding cavity of TLN, as the
prototype model of Zn21 metallopeptidases. This
cavity was built out of 53 amino acids, belonging to
the following oligopeptide segments:
Asn111-Trp115; Thr129-His146; Gly154-Asn159;
Asn165-Ile17l; Glu187-Val192; Leu202-Ser204; and
Gln225-Ile232. The coordinates of the main-chain
atoms are those of the X-ray structure of its complex
with thiorphan.12b The conformations of the
sidechains are those resulting from energy-minimiza-
tion of this TLN complex with thiorphan, in the
course of an ongoing study of the comparative bind-
ing energies of a series of thiolate inhibitors to TLN
(Tiraboschi, G. et al., work in progress). The protein
modulates the binding energy preferences in a man-
ner which is difficult to predict without direct numeri-
cal tools. We hope to also be able to make predictions
of numerical values for some other 2X to 2Y func-
tional group exchanges, which can be tested experi-
mentally to check on the behavior of the force
field–continuum model combination. The total inter-
molecular energy DE includes, in addition to the
interaction energies of the inhibitors with TLN, the
mutual side chain–side chain interactions as well.
The three amino acids binding Zn21 are His142,
His146, and Glu166. Each of the three anions was
docked by energy-minimization of the six intermo-
lecular variables defining its orientation. A single-
point computation was then done on the energy-
minimized structure, now including the continuum
solvation energy.
To complete the energy balances, it is necessary to
determine the TLN energy in the absence of bound
inhibitors. In the ‘‘uncomplexed’’ TLN, the presence
of a bound water molecule was detected, with an -Zn
distance of 1.88 Å.38 Such a distance is, however,
larger by 0.25 Å than those found in the Zn-water
complexes within Zn21 oligoligates investigated
above. It is, on the other hand, similar to the
distances actually found in Zn21 complexes with
OH- in a series of its oligoligates having OH- in its
first coordination shell (D.W. Deerfield, private com-
munication; Gresh, N., work in progress). This led us
to use OH- instead of H2O as the fourth Zn21 ligand
within the TLN cavity to define the initial state of
TLN in the absence of inhibitors. Such an assump-
tion is, furthermore, supported by the computations
of Jacob and Giessner-Prettre,39 showing a more
favorable energy balance with a water deprotonated
and Glu143 protonated than the converse state. In
the energy-minimized TLN-X complex, Zn21 is bi-
dentate-bound to X, at equal distances of 2.02 Å. The
C-bound O is H-bonded to the doubly protonated
His231 (dO-N 5 2.7 Å), and the N-bound O is
H-bonded to the protonated Glu143 residue (dO-O 5
3.5 Å). These features are similar to those observed
in the X-ray structure of the TLN complex with a
hydroxamate inhibitor.14 On the other hand, the
N-bound hydrogen is H-bonded to the main-chain O
of Ala113, instead of the sidechain of Glu143, as in
the X-ray structure. A stereo representation of the
complex is shown in Figure 8.
In the energy-minimized TLN-P complex, both
anionic oxygens are bound to Zn21, with distances of
2.11 and 2.38 Å. One anionic O is H-bonded to
His231 (dO-N 5 2.62 Å) and the other is H-bonded to
Glu143 (dO-O 5 2.88 Å). The amide N is H-bonded to
the main-chain O of Phe114 (dN-O 5 3.15 Å). In the
energy-minimized TLN-S complex, the S-Zn dis-
tance is 2.44 Å, close to the experimental distance of
2.38 Å. In the energy-minimized TLN-OH(-) com-
plex, the O-Zn distance is 1.88 Å, as in the X-ray
structure (without prejudging on the protonation
state of water). The onset of additional H-bond
distances in the X and P-TLN complexes results in
improved energy balances with respect to S, as
 MODELING OF INHIBITOR–METALLOENZYME INTERACTIONS 57

Fig. 8. Stereoview of the TLN–methylhydroxamate complex.

shown in the following energy balances: nents of the response of the solvent to the solute
potential).
TLN com- OH(2) X P S These energy balances give the X complex as
plex with having a stability identical with that of the OH(-) one
DE 2963.2 2970.6 2958.0 2939.6 used to define the ‘‘uncomplexed’’ state of TLN. The
DHhydr 21476.2 21449.6 21455.5 21457.6
complex with P is slightly more stable, whereas the S
DE 1 DHhydr 22439.4 22420.2 22413.5 22397.2 one is the least stable. A finer assessment would
require one to relax the sidechains of the TLN
DEsolv 2126.8 2107.6 298.0 292.0 residues as well (as done in Ref. 33), as well as the
desolvation position of Zn21. More important, it will request to
energy explicitly take into account, and energetically con-
front, a variety of inhibitors having X, P, or S as their
dE 5 DE 1 22312.6 22312.6 22315.5 22305.2
anionic moieties. This is the objective of ongoing
DHhydr 1
DEsolv calculations in one of our laboratories, which are
d, difference of 0.0 0.0 22.9 7.4 devoted to a series of TLN inhibitors (Tiraboschi, G.,
energy bal- et al., work in progress).
ances with Experimentally, the best TLN inhibitors are found
respect to in the phosphoramidate series. This occurs in spite of
OH(2) the intrinsically greater affinity of X than P for
d, difference of 2.9 2.9 0.0 10.3
Zn21, as well as the much better energy balances in
energy bal-
ance with
Zn-HHEX than in Zn-HHEP. The ‘‘equalizing’’ effect
respect to P of the protein on the affinities of X and P (actually
slightly better for P), seems consistent with this fact.
In contrast to the model oligoligates, the ‘‘protein A Ki value of 0.015 µmol was reported for P-Leu-Phe-
effect’’ has equalized the DHhydr energies of the P and OH.40 Ki values that are one order of magnitude
S complexes and diminished that of the X one. This is larger were found in the methylhydroxamate and
due to the greater inaccessibility of the anions in the methanethiolate series, translating a reduced bind-
TLN cavity, as contrasted to the model complexes. ing affinity. Thus a value of 0.4 µmol was reported for
The interplay of solvation/desolvation effects, along a benzyl-malonyl-L-Ala-Gly-p-nitroanilide hydroxa-
with the onset of additional H-bonds with TLN, mate derivative,14a and a value of 0.75 µmol was
yielded energy balances that differ by at most 10.4 reported for a (2-benzyl-3-mercaptopropanoyl)-L-
kcal/mol, despite the considerably magnified values alanylglycinamide mercaptan derivative.12a Further-
of DE and DHhydr with respect to their values in the more, these experimental studies have shown the
model tetra- and hexaligates (we note that the net binding affinities of inhibitors to be very sensitive to
charge in the four TLN complexes is 24, instead of 0 the integral structure of the molecule, whose anionic
in the model complexes, which accounts for the moiety acts as an anchoring point. This is exempli-
enhancement of DHhydr, which in the continuum fied by the phosphoramidate series, in which varia-
procedure is governed by the electrostatic compo- tions by up to three orders of magnitudes in the
58 D.R. GARMER ET AL.
binding constants were observed upon increasing
the hydrophobic character of the sidechains.40 Simi-
lar results were reported in the hydroxamate
series.14a This again highlights the other facet of the
forces at work in inhibitor–metallopeptidase interac-
tions, concurrently to ionic and H-bond interactions,
namely, hydrophobic interactions. The present study
has shown the capability of SIBFA to deal with ionic
interactions in oligoligated complexes. Its capability
to deal with H-bonds and the cooperative character
in multiply H-bonded complexes was also recently
shown by one of us in a joint SIBFA/ab initio study.9h
How accurately hydrophobic interactions can be
evaluated using the continuum model is a question
which must await further calculations on a variety of
inhibitors.
CONCLUSIONS
The results of comparing SIBFA calculations and
quantum chemistry on anionic inhibitor-protein mod-
els shows that the force-field method is capable of
calculating accurate structural data very rapidly for
trial binding structures. For example, this force-field
model produces model inhibitor coordination struc-
tures which are in good agreement with crystallo-
graphic data. By contrast, simulations using stan-
dard molecular mechanics resulted in structural
differences from experimental results, including an
increase in the zinc coordination number for thermo-
lysin.32 The advantage of SIBFA is its ability to
reproduce benchmark ab initio binding energies to
approximately 2% precision. This required a detailed
representation of the important nonadditive polariza-
tion and charge transfer energies, which vary widely
depending on the structure of the complete coordina-
tion shell.
The potency of various molecules as enzyme inhibi-
tors can then be approximately evaluated from the
energetics of interaction, including the contribution
of solvent from a continuum model. For example, the
relative binding preference of the inhibitors was
evaluated as methylhydroxamate . methanethi-
olate . methylphosphoramidate. In a previous study
using the integrated SIBFA/continuum procedure,
we successfully interpreted the effect of active site
mutation in thermolysin on the relative binding
strength of inhibitors bearing a thiolate or a phos-
phoramidate coordinating group.33 This could open
the way toward a rapid evaluation of inhibitor
docking. The model is also able to reproduce poten-
tial energy surfaces and energy differences for re-
lated structures. This makes SIBFA a less time-
consuming alternative to quantum chemistry
techniques for theoretical studies of exchange reac-
tions and modeling of transition state–protein inter-
actions. In addition to these, several other applica-
tions are planned, such as to supramolecular
chemistry,41 cation–ionophore complexes, ionic clus-
ters, and condensed phases involving ionic interac-
tions, etc.
Completion of the energy balances in the model
Zn21 and Mg21 tetra- and hexaligates, by taking
into account the continuum solvation/desolvation
effects, resulted in absolute values of DH’s all inferior
to 25 kcal/mol. The most favorably bound Zn complex
is the pentacoordinated arrangement Zn-HHEX, hav-
ing two histidines, one formate, and one methylhy-
droxamate anion. The least favorably bound one is
the hexacoordinated arrangement having two carbox-
ylate, two carbonyl, and two water oxygen ligands.
Their binding affinities differ by 11 kcal/mol. Dock-
ing of the methylhydroxamate, methanethiolate,
methylphosphoramidate, as well as the hydroxy
anions, in a binding site of TLN made out of 53
residues led to a more accented equilibration of the
energy balances for their binding. Thus, the best-
bound anion, methylphosphoramidate, and the least
favorably bound one, methanethiolate, had energy
balances differing by a smaller amount, 10.3 kcal/
mol, despite the considerable increase in the magni-
tudes of the individual terms, and despite the limited
flexibility allowed in the present minimizations.
This indicates that a very important contribution to
the modulation of the relative affinities of competing
inhibitors should originate from the H-bonds and
hydrophobic interactions contributed by the rest of
the inhibitors’ structure.
Finally, we consider the implications of the penul-
timate section of results for the application of the
hard-and-soft acids-and-bases principle. HSAB
trends have been disparaged as being practically
useless for predicting biological interactions.17 Our
more specific results show that HSAB is limited to
making only certain types of predictions. Specifi-
cally, the electronic selectivity for a soft dication
Zn21 over Mg21 occurs readily for binding sites
with soft ligands such as RS- donors. However, there
is no inherent selectivity of binding sites composed of
hard ligands toward Mg21 from electronic energies,
even though hydration energies for Zn21 have
greater magnitude. This is because the interaction
terms other than Ect and Ecorr do not appear to
systematically contribute large selectivity factors for
cations of similar size and charge. This is adverse to
the conventional view that hard–hard pairs are
favored by ionic interactions. Thus, occupation for
hard-ligand binding sites can only be controlled by
size constraints, ion concentrations, or irreversible
kinetic processes.
Also, it is likely that HSAB fails when considering
ions of different charge because of the wide variation
in electrostatic interactions which can occur with the
ligands. Thus, HSAB is a useful theoretical concept
for condensed phase and biological problems, but
should not be pushed to make predictions that
actually require modeling and geometric concepts to
understand them properly. SIBFA is as a good method
 MODELING OF INHIBITOR–METALLOENZYME INTERACTIONS
for this goal because it appears to reproduce the
selectivity results from high-level quantum chemis-
try with an encouraging level of precision.
ACKNOWLEDGMENTS
We thank the Institut du Développement et des
Ressources en Informatique Scientifique, Orsay,
France, on which part of the ab initio computations
were carried out, and Dr. Marie-Claude Fournié-
Zaluski for reviewing the manuscript.
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