Hydrolytic catalysis and structural stabilization in a designed metalloprotein

Article abstract of DOI:10.1038/nchem.1201

Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions. Reproducing these functions in a designed protein is the ultimate challenge to our understanding of them. Here, we present an artif

Full text of DOI:10.1038/nchem.1201

ARTICLES
PUBLISHED ONLINE: 27 NOVEMBER 2011 | DOI: 10.1038/NCHEM.1201
Hydrolytic catalysis and structural stabilization in a
designed metalloprotein
1
1†
2
1
Melissa L. Zastrow , Anna F. A. Peacock , Jeanne A. Stuckey and Vincent L. Pecoraro
*
Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions.
Reproducing these functions in a designed protein is the ultimate challenge to our understanding of them. Here, we present
an artificial metallohydrolase, which has been shown by X-ray crystallography to contain two different metal ions—a Zn(II
)
ion, which is important for catalytic activity, and a Hg(II) ion, which provides structural stability. This metallohydrolase
displays catalytic activity that compares well with several characteristic reactions of natural enzymes. It catalyses
p-nitrophenyl acetate (pNPA) hydrolysis with an efficiency only ∼100-fold less than that of human carbonic anhydrase
(
CA)II and at least 550-fold better than comparable synthetic complexes. Similarly, CO hydration occurs with an efficiency
2
within ∼500-fold of CAII. Although histidine residues in the absence of Zn(II) exhibit pNPA hydrolysis, miniscule
apopeptide activity is observed for CO hydration. The kinetic and structural analysis of this first de novo designed
2
hydrolytic metalloenzyme reveals necessary design features for future metalloenzymes containing one or more metals.
etal ions are found in over one-third of natural proteins
Our designed metallopeptides represent a middle ground
and are essential to many biological processes, including between studying the native metalloprotein and preparing small-
hydrolytic chemistry, respiration and photosynthesis. molecule synthetic models. In fact, they offer a number of advan-
M
These elements make up various sites, one or more in a single tages not easily found in small molecules, including the ability to
1
protein, with catalytic, structural and electron transfer functions . work under aqueous conditions, the use of natural amino-acid
One of the most challenging approaches to understanding how ligands, and stabilization of lower coordination numbers within a
metals function in proteins is to reproduce these sites using hydrophobic core. In comparison to natural proteins, our constructs
de novo protein design. The successful preparation of artificial metallo- are significantly simplified to retain only core elements, allowing us
enzymes not only allows one to reveal previously unappreciated to isolate and establish different contributing factors to protein
features defining metal behaviour in biology, but also inspires con- function. Furthermore, these peptides can be designed for increased
fidence in the ability to design novel systems with catalytic reactivity thermal stability, and their facile synthesis allows the incorporation
9
not naturally observed that could support a wide range of biotech- of non-coding amino acids .
2,3
nological and pharmaceutical applications . Although considerable
Our objective was to incorporate a catalytically active mono-
progress has been made in enhancing the catalytic properties of nuclear metal site into a de novo designed construct and, at the
natural enzymes through protein redesign methods, there are few same time, incorporate a separate and distinct structural site for
2–5
examples of successful de novo designed metalloenzymes . Our stabilization. This represents both the first example of a de novo
bottom-up’ approach is to design a three-stranded coiled coil designed mononuclear metalloenzyme and of a de novo metallopro-
‘
(
3SCC) containing both a catalytic metal site, ZnN O, as found in tein containing two different metals in two different coordination
3
carbonic anhydrase (CA), and a separate HgS site for structural environments with distinct functions. We targeted CA for our
3
6,7
stabilization . Our 3SCCs are simplified constructs that retain active site because it displays high catalytic activity. Conveniently,
sufficient complexity to resemble a native protein environment. its threefold symmetric histidine (His) binding site fits well into
There has been significant interest recently in the design of arti- the designed coiled coil scaffolds, and its numerous studies
ficial metalloenzymes, with two fundamentally different approaches provide us with a wealth of literature to which we can compare
being applied. The first involves introducing a metal site into a pre- our results. Previous reports exist on the design of Zn(His) O
3
1
0–12
existing, stable natural protein fold. The second requires both the sites into various de novo protein folds
and into stable natural
1
3,14
design of a catalytic metal site and a sequence that folds into a protein structures ; however, neither the structural elucidation
8
defined three-dimensional structure . This approach exploits and of these centres nor their catalytic activity has been reported. Our
tests first-principle understanding of biophysics and metallobio- catalytically active ZnN O-type site therefore represents the first
3
chemistry with the ultimate goal of establishing criteria that allow structurally characterized synthetic and active CA site within a
for the generation of stable new catalysts presently not found in ‘protein framework’.
biology. Unfortunately, there are few reports of true de novo metallo-
2
–5
enzymes based on this second approach, and none are hydrolytic
.
Results and discussion
One elegant example has been reported in which the active site con- Design of stabilizing structural and catalytic sites. We have
sists of a di-iron centre in the interior of a four-helix bundle that is reported numerous studies investigating the binding of metal ions
4
,5
capable of catalysing the O -dependent phenol oxidase reaction . such as Hg(II), Cd(II), As(III), Bi(III) and Pb(II) to 3SCC scaffolds
2
The authors remarked on the important balance between confor- based on the TRI family of peptides, Ac-G(LKALEEK) G-NH ,
4
2
mational stability and catalytic activity.
which contain thiol substitutions for leucine (Leu) residues in the
1
2
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan
†
4
8109, USA, Present address: School of Chemistry, University of Birmingham, Edgbaston B15 2TT, UK. *e-mail: vlpec@umich.edu
118
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a
c
b
2
n1
3
Figure 1 | Ribbon diagrams of the [Hg(II)] [Zn(II)(H O/OH )] (CSL9PenL23H)
parallel 3SCC (one of two different three-helix bundles present in the
S
2
N
asymmetric unit) at pH 8.5. Shown are the main chain atoms represented as helical ribbons (cyan) and the Pen and His side chains in stick form (sulfur ¼
yellow; nitrogen ¼ blue; oxygen ¼ red). a, One of two trimers found in the asymmetric unit of the crystal structure. b, Top-down view of the structural trigonal
1
7
thiolate site, Hg(II)S , confirming the proposed structure of Hg(II) in Cys-containing TRI peptides . This metal site should mimic well the structural site in the
3
47
metalloregulatory protein MerR . c, Side view of the tetrahedral catalytic site, Zn(II)N O, which closely mimics carbonic anhydrase and matrix
3
1
metalloproteinase active sites . All figures are shown with 2F –F electron density contoured at 1.5s overlaid.
o
c
interior, and we have observed that the addition of metal ions to with negative minima at 208 and 222 nm. Molar ellipticity (u) values
these destabilized constructs confers significant stability ,7,15–17. obtained at 222 nm and pH 8.5 of 224,1028 dmol cm for
6
21
2
21
2
The peptides used in this study (see Supplementary Table S1 for (TRIL23H)3 and 225,7048 dmol cm for (TRIL9CL23H)3 are
full sequences) each contain an additional His for Leu consistent with folded coiled coil structures. Although these data
substitution in the interior of the 3SCC, which is expected to cannot be quantitatively fit to a two-state model (Supplementary
destabilize the peptide aggregate markedly. TRIL9CL23H contains Fig. S1), it is apparent that the midpoint for unfolding of the
both a His substitution in the 23rd position towards the doubly substituted (TRIL9CL23H) is at a lower concentration of
3
C-terminus and a thiol substitution for preparation of a structural denaturant than for (TRIL23H)3. The lower stability of
site in the 9th position, near the N-terminus (TRIL23H has only (TRIL9CL23H)3 is anticipated, as an additional hydrophobic
a Leu to His substitution at the 23rd position). When Hg(II) is packing layer has been substituted. As expected, a significant shift of
bound to the three cysteine (Cys) residues, this peptide will be the midpoint to a higher concentration is observed in the
2
2
referred to as [Hg(II) ](TRIL9CL23H)3 , where subscript ‘S’ denaturation for [Hg(II) ](TRIL9CL23H)3
, incorporating the
S
S
represents binding to the sulfur site. When Zn(II) is bound to the structural HgS3 site, as compared to that for both apopeptides
His site, there are three coordinated nitrogen atoms from protein (Supplementary Fig. S1). This shift clearly demonstrates the benefit
ligands and one exogenous water or hydroxide, depending on pH. of incorporating a structural Hg(II)S site into the design. UV-vis
3
2
We will use ZnN to indicate that the Zn(II) is bound at the His site spectra of [Hg(II) ](TRIL9CL23H)3 confirm that Hg(II) binds in a
S
and specify within parentheses the protonation state of the trigonal fashion in the sulfur site as compared to previously reported
1
8
coordinated solvent. Under pH conditions where the solvent may values for Hg(II) binding to Cys sites in the TRI peptide family .
3
2
2
occur both as the H O and OH forms, we will use H O/OH .
Protein crystallography clarifies how Hg(II) and Zn(II) bind to
the structural Cys and catalytic His sites, respectively. For these
2
2
þ
Hg(II)] [Zn(II)(H O)] (TRIL9CL23H)
3
[
will then refer to a
S
2
N
trigonally coordinated Hg(II) at the Cys site, and a pseudotetrahedral studies, a derivative of the analogous CoilSer (CS) peptide was used,
Zn(II) with three coordinated imidazoles and one water molecule.
CSL9PenL23H (Pen ¼ penicillamine, sequence Ac-E WEALEKK
PenAALESK LQALEKK HEALEHG-NH ); CoilSer behaves
2
Characterization of structural and catalytic sites. First we assessed very similarly to the TRI peptide family, but is more amenable to
9
,19
the utility of the proposed structural site, HgS , in TRIL9CL23H. To crystallographic characterization . Crystals in space group P21
3
determine the relative folding and stability of the 3SCC in the presence were obtained and the structure was solved at a resolution of
and absence of Hg(II), we monitored the peptide region of the circular 2.20 Å using molecular2 replacement. The resulting model,
nþ
dichroism (CD) spectrum that yielded characteristic coiled coil peaks [Hg(II)] [Zn(II)(H O/OH )] (CSL9PenL23H)
3
(pdb 3PBJ),
S
2
N
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1201
ARTICLES
determine if this strictly first-coordination sphere structural
relationship would be enough to confer catalytic activity. The
a-CAs can hydrolyse esters such as p-nitrophenyl acetate (pNPA)
by a similar mechanism (nucleophilic attack by Zn(II)-bound
hydroxide) to the physiologically relevant CO2 hydration
2
0,21
reaction . Modelling the structure and reactivity of this
metalloenzyme has been the focus of many studies; however,
relatively few active model complexes that can be considered
structurally true to the active site of CA have been reported for
2
2–27
pNPA hydrolysis
. Of these, there are three notable small-
molecule examples of N -type ligands reported to bind Zn(II) and
3
facilitate the hydrolysis of pNPA with measurable second-order
rate constants. These are the macrocyclic amine complexes
[
12]aneN3 and [15]aneN O , and a tris(imidazolyl)phosphine
3
2
2
1
2
21
complex. A second-order rate constant of 0.041 M
reported for [12]aneN at pH 8.2 and 25 8C (pK 7.3) and of
0
s
was
2
3
a
2
1 21
.6 M
s
for [15]aneN O , assuming 100% active hydroxide
3 2
2
5
complex (pK 8.8) . The Zn(II) complex of tris(4,5-di-n-propyl-2-
a
imidazolyl)phosphine is reported to catalyse this reaction in
2
1 21
micelles with a second-order rate constant of 0.86 M
s ;
however, the corresponding estimated value under aqueous (and
2
1 21
more comparable) conditions is only 0.0186 M
s
(ref. 24).
2
Figure 2 | Overlay of the Zn(II)N O site in [Hg(II)] [Zn(II)(H O/OH )]
3
S
2
N
There are also several relatively poor structural models, such as
n1
(
CSL9PenL23H)3 with the active site of human CAII. [Hg(II)]
S
[12]aneN4 with four coordinating nitrogens, which have rate
2
nþ
[Zn(II)(H O/OH )] (CSL9PenL23H)
is shown in cyan (pdb 3PBJ)
21 21
2
N
3
constants up to 0.1 M
(
s
at pH 9.3 (pK 7.9) in 10% CH CN
a
3
and CAII in tan (pdb 2CBA). The solvent molecule associated with
21 21
ref. 28) and up to 5.0 M
s
under micellar conditions (for the
2
nþ
[
Hg(II)] [Zn(II)(H O/OH )] (CSL9PenL23H)
is shown in red and that
29
S
2
N
3
1-hexadecyl derivative of [12]aneN ) . Notably, these synthetic
4
associated with CAII lies underneath. The model displays an excellent
structural overlay for the first coordination sphere atoms with CAII; however,
the orientation of the imidazoles differs between the two proteins. Another
subtle difference is that the present structure has three 1 amino nitrogens
bound to the Zn(II) ion, whereas CAII has a mixed two 1 and one d
coordination sphere. Hence, the present structure better mimics the matrix
metalloproteinase adamalysin II, which also has three 1 amino nitrogens
complexes, in addition to having important drawbacks such as
not being able to perform in 100% aqueous solution and often
dimerizing, typically suffer from product inhibition.
2
nþ
We find that [Hg(II)] [Zn(II)(H O/OH )] (TRIL9CL23H)
3
S
2
N
exhibits saturation kinetics for pNPA hydrolysis in buffered
23 21
aqueous solution, with k ¼ 2.2(+0.5) × 10
cat
s
and k /K ¼
cat
M
21 21
1
.38+0.04 M
s
at pH 7.5 (25 8C, 10 mM catalyst, turnover
4
8
bound to Zn(II) . Overlay was performed manually in Pymol. See
Supplementary Fig. S2 for a side-on view.
number (TON) . 10, Table 1). Compared to the best small-
molecule models (with mixed solvent systems), this designed metallo-
peptide is over 33-fold superior to [12]aneN (pH 8.2, TON , 1
3
contains two independent well-folded, parallel 3SCCs in the under the conditions at which this rate was measured) at its
asymmetric unit (Fig. 1a). One contains Hg(II) bound to the slowest (pH 7.5) and over 550-fold better at pH 9.5 (ref. 22).
sulfurs of Pen in a trigonal planar structure (Fig. 1b) with Hg(II)–S At pH 9.5, it is ꢀ40-fold faster than the maximal rate for
distances 2.21, 2.07 and 2.41 Å, and four-coordinate zinc bound [15]aneN O (ref. 25) and 1,250-fold faster than Brown’s phosphine
3
2
2
4
to three His ligands and a chloride ion. The second 3SCC contains complex under comparable conditions . Additionally, designed pro-
a T-shaped Hg(II) (with Hg(II)–S distances of 2.29, 2.13 and 3.06 Å) teins have been reported that can catalyse this reaction using His resi-
and a four-coordinate Zn(II) with three His residues and a water/ dues. Their maximal activities (at pH 7.0) are tenfold lower than the
2
hydroxyl ligand (Fig. 1c). The Zn(II)–His distances in both 3SCCs maximal [Hg(II)] [Zn(II)(OH )] (TRIL9CL23H) activity at pH 9.5
S
N
3
3
0–32
are close to 2.0 Å. Excitingly, the Zn(II)His X site closely resembles (see Supplementary Table S4 and Supplementary Discussion)
.
3
that found in CA (pdb 2CBA, Fig. 2) and is the best reported struc- The observed increase in rate and catalytic efficiencies of
2
nþ
[Hg(II)] [Zn(II)(H O/OH )] (TRIL9CL23H)
3
tural model for CA in a synthetic system.
as a function of
increasing pH suggests a change in the active site species, presumably
Characterization of ester hydrolysis. After demonstrating that we through deprotonation of the Zn(II)–aqua ligand. k /K of up to
S
2
N
cat
M
2
1 21
obtained the best structural model of CA, it was necessary to 23.3+0.3 M
s
can be reached at pH 9.5, whereas the KM values
2 n1
Table 1 | Kinetics for hydrolysis of p-nitrophenyl acetate by [Hg(II)] [Zn(II)(H O/OH )] (TRIL9CL23H) compared to CAII.
S
2
N
3
Designed metalloprotein (pK 5 8.8)*
CAII (pK 5 6.8)
a
a
21
21
21
21 21
s
21 †
)
pH
kcat/KM (M
s
)
kcat (s
)
KM (mM)
kcat/KM (M
)
kcat (s
KM (mM)
†
†
7
n.d.
1.38+0.04
3.1+0.1
6.0+0.1
10.8+0.3
17.6+0.3
23.3+0.3
n.d.
n.d.
1,670
37+10
22.1+5.0
23
23
23
23
23
23
‡
‡
7.5
2.2(+0.5) × 10
5.4(+1.5) ×10
12(+4) ×10
1.6+0.4
1.7+0.5
1.9+0.6
2.0+0.9
2.1+0.6
1.7+0.5
2,600+5
30.53+2.10
†
†
8
8
8
9
9
2,550
53+10
20.7
.5
.75
21(+10) ×10
38(+10) ×10
40(+12) ×10
†
†
2,320
56+10
23.9
.5
2
nþ
Conditions: 10 mM [Hg(II)] [Zn(II)(H O/OH )] (TRIL9CL23H)3 in 50 mM buffer (HEPES or CHES). kcat, KM and kcat/KM calculations and their error analysis are described in the Supplementary Methods.
S 2 N
‡
*
†
From ref. 20. From ref. 33 at pH 7.4.
120
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a
b
2
2
1
1
5
0
5
0
5
0
0.05
0.04
0.03
0.02
0.01
0.00
7.5
8.0
8.5
pH
9.0
9.5
7.5
8.0
8.5
pH
9.0
9.5
2
n1
Figure 3 | pH-dependency of pNPA hydrolysis by [Hg(II)] [Zn(II)(H O/OH )] (TRIL9CL23H) . a,b, Plots of kcat/KM versus pH (a) and kcat versus pH (b)
S
2
N
3
2
nþ
for the hydrolysis of pNPA by [Hg(II)] [Zn(II)(H O/OH )] (TRIL9CL23H)
(10 mM). pK values of 8.82+0.11 and 8.77+0.08 for a and b, respectively,
a
S
2
N
3
2
can be determined from the fittings and presumably represent the deprotonation of Zn–OH to form an active Zn–OH nucleophile, as with CAII. The bars
2
shown represent the goodness of linear fits to the Lineweaver-Burke (double reciprocal) plots of initial rate data for a range of substrate concentrations at
each pH (replicate trials for each initial rate were averaged prior to those fits). Note that kcat errors are relatively large because solubility limitations of the
substrate precluded our ability to test substrate concentrations at or above the KM values. See Supplementary Methods for a description of the fitting and
error analysis.
remain essentially constant (ꢀ2 mM, within error) over the range. site enhances stability with no expense to catalytic activity.
2
pH-dependency profiles can be fitted to yield pK values of 8.82+ Furthermore, above pH 9.0, [Hg(II)] [Zn(II)(OH )] (TRIL9CL23H)
a
S
N
3
0
.11 for k /KM versus pH and 8.77+0.08 for kcat versus pH, exhibits ꢀ20% enhanced catalytic efficiency compared to the
cat
suggesting that the rate-limiting step is chemical in nature complex lacking the structural site, suggesting the added stability con-
(
[
Fig. 3). Notably, variation of the concentration of ferred by Hg(II) may be important at higher values of pH.
2
Hg(II)] [Zn(II)(OH )] (TRIL9CL23H) relative to substrate at
S
N
3
pH 9.5 confirms that the reaction is first order in the Zn(II) Evaluation of CO hydration activity. Of course, CO hydration is
2
2
protein concentration over the tested range of 10–60 mM catalyst. the native reaction catalysed by CA, so we have evaluated whether our
Furthermore, we do not observe product inhibition under the con- complex can perform this chemistry. This is a relatively facile reaction,
ditions of our kinetic experiments (for p-nitrophenol or acetate up especially for model complexes, because product inhibition is less
to 15 mM and at a single substrate concentration of 0.5 mM). A of a problem than for pNPA hydrolysis. Although there are many
3
6–43
detailed analysis with potassium acetate (varying both pNPA and examples of Zn(II) complexes that can catalyse CO hydration
,
2
acetate concentrations) yields competitive inhibition with a rather the macrocyclic amine [12]aneN and Brown’s phosphine complex
4
high KI of 0.45 M. (See Supplementary Table S4 and (described earlier in terms of pNPA hydrolysis) are among the
4
0,42
Supplementary Discussion for the above comparisons of catalytic fastest models . The fastest N -type model, therefore, is tris(4,5-
3
efficiencies and second-order rate constants as well as those of the di-n-propyl-2-imidazolyl)phosphine, with a reported second-order
21 21
peptides in the absence of Zn(II).) Given that imidazole is known rate constant of 2,480 M
s . CAII can catalyse this reaction with
to catalyse this reaction, the apopeptide (containing three imida- a pH-independent turnover per second of approximately one
5
21
zoles in the trimer) also facilitates hydrolysis with an efficiency at million (8.2 × 10 s
)
and
a
catalytic efficiency of 9.2 ×
7
21 21
pH 9.0 that represents ꢀ30% of that of the Zn(II)-bound trimer 10 M
s , which is nearly 30,000-fold faster than even
this activity is accounted for in the values reported for Zn(II)-facili- [12]aneN4 and 37,000-fold over the more structurally faithful
tated hydrolysis). Notably, little to no background peptide activity is tris(4,5-di-n-propyl-2-imidazolyl)phosphine complex
(
4
0,42,44
.
We
observed for CO hydration (below). have performed CO hydration studies at pH 9.5 and 25 8C. We
2
2
2
The maximum k /K achieved for pNPA hydrolysis by find that [Hg(II)] [Zn(II)(OH )] (TRIL9CL23H) attains a k of
cat
M
S
N
3
21 21
s
cat
2
3
5
[
Hg(II)] [Zn(II)(OH )] (TRIL9CL23H) is just over 100-fold less at 1.8(+0.4)×10 , a k /K of 1.8(+0.3)×10 M
and a KM of
S
N
3
cat
M
pH 9.5 than that of CAII at pH 8 (maximum efficiency). 10.0+2.4 mM. This catalytic efficiency is over 70-fold faster than
20,33
Furthermore, it is only ꢀ16-fold less efficient than CAI . Notably, any previously reported model and within ꢀ500-fold of CA II.
secondary interactions in CA (as in many metalloenzymes) are very Future work will involve not only completing the determination of
important; in particular, mutation of Thr199 (which forms a hydro- this activity over the full pH range (7.5–9.5), but will also
gen-bonding interaction with the hydroxide nucleophile of the Zn(II) incorporate potential hydrogen-bonding residues in an attempt to
centre) in CAII results in a 100-fold loss in catalytic efficiency and a improve the properties of our model.
1
.5–2.5 unit increase in pK from 6.8 (refs 34,35). These new kinetic
a
parameters for T199A CAII would yield an enzyme with an efficiency Conclusions
2
within a factor of two of [Hg(II)] [Zn(II)(OH )] (TRIL9CL23H) and The design reported here takes advantage of our ability to generate
S
N
3
an essentially identical pK . Thus, the catalytic efficiency of our peptides that contain adjacent metal ion sites with differing affi-
a
4
5,46
minimal first-generation metallopeptide model towards pNPA nities, properties and functions . We have prepared two spatially
hydrolysis, which has yet to take into account these secondary inter- removed sites within the interior of a coiled coil, in which very
actions that significantly contribute to optimal activity in CA, is essen- different coordinating ligands are presented to the metal ions.
tially mimicking one of the fastest known enzymes, CAII. It should be These sites differ greatly and we are now capable, for the first
2þ
noted that the relative k /K values for [Zn(II)] (TRIL23H)3 are time, of building an artificial protein that contains both structural
cat
M
N
similar/identical from pH 7.5 to 8.75, suggesting that the structural and catalytic sites. Not only is this the first example of a true
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de novo designed protein containing two separate metal sites with
different functions, but it is also the first hydrolytic metalloenzyme
designed from scratch that is competitive with one of the fastest
known natural metalloenzymes, CA. Furthermore, it represents a
unique synthetic model with rates higher than any previously
designed three-stranded coiled coils. Proc. Natl Acad. Sci. USA 104,
11969–11974 (2007).
2
2
0. Verpoorte, J. A., Mehta, S. & Edsall, J. T. Esterase activities of human carbonic
anhydrases B and C. J. Biol. Chem. 242, 4221–4229 (1967).
1. Gould, S. M. & Tawfik, D. S. Directed evolution of the promiscuous esterase
activity of carbonic anhydrase II. Biochemistry 44, 5444–5452 (2005).
reported nitrogenous catalytic Zn(II) complex for either pNPA 22. Kimura, E., Shiota, T., Koike, T., Shiro, M. & Kodama, M. A Zinc(II) complex
of 1,5,9-triazacyclododecane ([12]aneN ) as a model for carbonic anhydrase.
J. Am. Chem. Soc. 112, 5805–5811 (1990).
3. Olmo, C. P., Bohmerle, K. & Vahrenkamp, H. Zinc enzyme modeling with zinc
complexes of polar pyrazolylborate ligands. Inorg. Chim. Acta 360,
hydrolysis or CO hydration. An important question in protein
3
2
design that is difficult, if not impossible, to address using mutagen-
esis studies on the natural protein in question, is what the minimal
unit required for catalytic activity is. We have begun to answer this
2
1510–1516 (2007).
question with our first coordination sphere-only model and hope to 24. Koerner, T. B. & Brown, R. S. The hydrolysis of an activated ester by a tris(4,5-di-
2
þ
gradually build in secondary interactions to gain further insight. The
versatility of the TRI system will allow us to readily make changes to
incorporate residues capable of secondary interactions like hydro-
gen bonding and to study the resulting effects on both catalytic
n-propyl-2-imidazolyl)phosphine–Zn complex in neutral micellar medium as
a model for carbonic anhydrase. Can. J. Chem. 80, 183–191 (2002).
2
5. Bazzicalupi, C. et al. Carboxy and phosphate esters cleavage with mono- and
dinuclear zinc(II) macrocyclic complexes in aqueous solution. Crystal structure
2
of [Zn L1(m-PP) (MeOH) ](ClO ) (L1¼[30]aneN O , PP ¼diphenyl
2
2
2
4 2
6 4
activity and pK . These results also inspire confidence that more
phosphate). Inorg. Chem. 36, 2784–2790 (1997).
a
economically important processes may be developed within 26. Sprigings, T. G. & Hall, D. C. A simple carbonic anhydrase model which achieves
catalytic hydrolysis by the formation of an ‘enzyme-substrate’-like complex.
a
biomolecular scaffold for future biotechnological and
J. Chem. Soc. Perkin Trans. 2, 2063–2067 (2001).
pharmaceutical applications.
2þ
2
2
7. Jairam, R., Potvin, P. G. & Balsky, S. Zn inclusion complexes of endodentate
tripodands as carbonic anhydrase-inspired artificial esterases. Part 2. Micellar
systems. J. Chem. Soc. Perkin Trans. 2, 363–367 (1999).
Methods
See Supplementary Information for additional experimental details.
8. Koike, T., Takamura, M. & Kimura, E. Role of zinc(II) in b-lactamase II: a model
study with a zinc(II)-macrocyclic tetraamine (1,4,7,10-tetraazacyclododecane,
cyclen) complex. J. Am. Chem. Soc. 116, 8443–8449 (1994).
29. Kimura, E., Hashimoto, H. & Koike, T. Hydrolysis of lipophilic esters catalyzed
by a zinc(II) complex of a long alkyl-pendant macrocyclic tetraamine in micellar
solution. J. Am. Chem. Soc. 118, 10963–10970 (1996).
30. Broo, K., Brive, L., Ahlberg, P. & Baltzer, L. Catalysis of hydrolysis and
transesterification reactions of p-nitrophenyl esters by a designed helix-loop-
helix dimer. J. Am. Chem. Soc. 119, 11362–11372 (1997).
Received 25 March 2011; accepted 18 October 2011;
published online 27 November 2011
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1201
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M.L.Z. carried out the experimental work and completed crystal structure refinement.
A.F.A.P. obtained and solved the crystal structure. M.L.Z., A.F.A.P., J.A.S. and V.L.P.
contributed to the design of the experiments and analysis of the data. All authors
contributed to writing the paper.
Additional information
Acknowledgements
The authors declare no competing financial interests. Supplementary information
accompanies this paper at www.nature.com/naturechemistry. Reprints and permission
information is available online at http://www.nature.com/reprints. Correspondence and
requests for materials should be addressed to V.L.P.
V.L.P. acknowledges support from the National Institutes of Health (grant no. R01 ES0
2236), and M.L.Z. from the National Institutes of Health Chemistry–Biology Interface
1
Training Program. Use of the Advanced Photon Source was supported by the
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2012 Macmillan Publishers Limited. All rights reserved.