Zn(ii) complex for selective and rapid scission of protein backbone

Article abstract of DOI:10.1039/b818022a

A Zn^II complex with an aldehyde group hydrolyzed porcine pancreatic elastase under mild conditions, pH 8.0, 50 °C, by the Schiff base formation between the Zn^II complex and the NH₂ group in the protein, suggesting that a Zn^II compound can be active toward peptide hydrolysis when it strongly binds to the substrate.

Full text of DOI:10.1039/b818022a

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Zn(II) complex for selective and rapid scission of protein backbonew
Morio Yashiro,*a Yukiko Kawakami,b Jun-ichi Taya,b Suguru Araib and Yuki Fujii*b
Received (in Cambridge, UK) 14th October 2008, Accepted 19th December 2008
First published as an Advance Article on the web 29th January 2009
DOI: 10.1039/b818022a
A ZnII complex with an aldehyde group hydrolyzed porcine
pancreatic elastase under mild conditions, pH 8.0, 50 1C, by the
Schiff base formation between the ZnII complex and the NH2
group in the protein, suggesting that a ZnII compound can be
active toward peptide hydrolysis when it strongly binds to the
substrate.
We now report that a ZnII complex having an aldehyde
group, ZnII-3, can hydrolyze a protein under near physio-
logical conditions, pH 8.0, 50 1C, suggesting that ZnII can be
very active toward peptide hydrolysis when it strongly binds to
the substrate polypeptide.
Suitable molecular design of a ZnII complex was searched
for on the basis of activity toward hydrolysis of GlyGly under
pH 7.0–9.5 at 70 1C: A buffered solution (N-2-hydroxyethyl-
piperazine-N0-2-ethanesulfonate (HEPES) 50 mM, pH 7.0,
8.1, 9.0 or 9.5) containing GlyGly (2.0 mM), the chelate ligand
(1, 2, 3 or 4, 2.0 mM) and ZnSO4 (2.0 mM) was heated at 70 1C
in a water bath, and the reaction was followed by HPLC.7,10
All reactions examined yielded Gly as the decomposition
product of GlyGly, indicating that hydrolysis of the peptide
bond was promoted by ZnII complexes. The pseudo-first-order
rate constants were summarized in Table 1.
Zinc is an essential element in living organisms. Enzymes such
as carboxypeptidase A, thermolysin, leucine aminopeptidase
and a series of matrix metalloproteinases require ZnII in the
catalytic center.1
Studies on the nonenzymatic hydrolysis of a protein back-
bone using a metal ion or complex, involving CuII,2 FeII,3
NiII,4 PdII,5 PtII5e or MoIV,6 have been carried out. However,
little success has been reported with any ZnII compounds.7
Accumulated knowledge regarding ZnII in enzymes tells us
that this element plays critical roles in promoting hydrolysis in
many peptidases,1,8 but we have not yet achieved as high a
level of chemistry as that of an enzyme. There still remains a
lot to reveal about the chemistry of ZnII in order to use its
potential ability as a catalyst in non-enzymatic systems.
ZnII is labile for the ligand exchange reaction. Chelation of
ZnII with glycine oligopeptides is reported to occur at the
amino terminus and the amide carbonyl oxygen with a stability
constant, logK, of 3.1.9 The relatively low stability constant of
ZnII compared with those of CuII or NiII indicates that only a
small fraction of ZnII binds to peptides at millimolar con-
centration ranges or below, and the coordination is very
sensitive to the environment, in which competitive species,
such as buffer molecules, are abundant. One of effective ways
to create a stable coordination of a ZnII complex with a
substrate peptide is to bind them using a covalent bond
between the chelate ligand of ZnII and the substrate. We have
tried to use an imine bond, or a Schiff base: a ZnII complex
having an aldehyde group will readily bond to an amino group
of a peptide, i.e. the N-terminus or side chains of lysine
residues, in aqueous solutions. The effectiveness of the Schiff
base formation between the metal chelate and the target
protein in the protein hydrolysis has been demonstrated using
CuII complex systems.2d,f
ZnII-1 and ZnII-2 showed greater activity toward peptide
hydrolysis than free ZnII at pH 7.0. 1 and 2 seemed to be
promising as a binding moiety for ZnII. Then, an aldehyde
group was introduced to 2 through N-substitution with
3-chloromethyl-5-methylsalicylaldehyde, resulting in a new
chelate ligand 3.
The Schiff base formation between GlyGly and the aldehyde
ligand 3 was clearly evidenced by 1H NMR spectroscopy
(Fig. S4w). The singlet at 9.75 ppm due to –CHQO dis-
appeared completely when GlyGly and ZnII-3 were mixed at
pD 6.2, 8.2 or 9.0. Instead, a singlet appeared at 8.04 ppm,
which can be ascribed to –CHQN–. The quantitative forma-
tion of the imine bond was strongly indicated. ZnII-3 was
the most active toward GlyGly hydrolysis at pH 8.1 and 9.0
among ZnII complexes examined in this study, indicating that
the Schiff base formation moderately increases the peptide
hydrolysis activity of the ZnII complex. For comparison,
ZnII-4 was tested, and found to be less active toward peptide
hydrolysis compared with ZnII-3 at both pH 7.0 and 9.0,
indicating an important role of the alcohol arm of 3 in peptide
hydrolysis.
a Department of Nanochemistry, Faculty of Engineering, Tokyo
Polytechnic University, Iiyama, 1583, Atsugi, 243-0297, Japan.
E-mail: yashiro@nano.t-kougei.ac.jp; Fax: (+81)46-242-9554;
Tel: (+81)46-242-9554
Then, the reaction of bovine serum albumin (BSA,
Fig. S5(a)w) with ZnII-2, ZnII-3 and ZnII-4 was examined.
BSA was found to be very slowly decomposed under weak
basic conditions, and high pH conditions were needed for
practical observation of the BSA hydrolysis. Thus, we
employed pH 11.0 for the hydrolysis experiment of BSA with
ZnII complexes. BSA (9.0 mM) and the ZnII complex
b Department of Material and Biological Science, Faculty of Science,
Ibaraki University, Bunkyo 2-1-1, Mito, 310-8512, Japan.
E-mail: yuki@mx.ibaraki.ac.jp; Fax: (+81)29-228-8403
w Electronic supplementary information (ESI) available: Supplementary
figures and experimental details. See DOI: 10.1039/b818022a
ꢀc
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Table 1 Pseudo-first-order rate constants (kobs) for GlyGly hydrolyses and BSA hydrolyses in the presence of ZnII complexes
obs/10ꢁ3 hꢁ1 for GlyGly hydrolysisa
k
kobs/10ꢁ3 hꢁ1 for BSA decomposition at pH 11.0b
pH 7.0
pH 8.1
pH 9.0
pH 9.5
Complex
None
0.099
1.1
1.2
1.4
0.45
0.13
0.17
0.61c
1.7
1.3
2.1
0.28
0.57c
2.0
2.4
3.8
—
—
—
—
4.6
1.1
5.6
13
—
16
44
23
ZnII ion
ZnII-1
ZnII-2
ZnII-3
ZnII-4
—
0.94
a
b
c
[GlyGly]0 = 2.0 mM, [ZnII complex] = 2.0 mM, at 70 1C. [BSA]0 = 9.0 mM, [ZnII complex] = 0.91 mM, pH 11.0 at 50 1C. Precipitates were
formed during the reaction.
(0.91 mM) were dissolved in a buffer (cyclohexylaminopropane-
sulfonate (CAPS) 50 mM, pH 11.0) and the mixture was heated
at 50 1C. The reaction was followed by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE). Analysis of
the intensity of the BSA band gave rate constants for the
decomposition, which were also summarized in Table 1. The
decomposition of BSA was indeed promoted by ZnII com-
pounds, and ZnII-3 was the most effective. The SDS-PAGE
analysis of the reaction with ZnII-3 showed many fragment
bands, e.g. 55, 50, 45, 39, 35, 31, 27, 18 and 16 kDa (Fig. S6w).
However, the intensities of these fragment bands were very low,
Fig. 1 SDS-PAGE analysis (10–20% gradient gel) of the reaction
mixture of porcine pancreatic elastase (62.4 mM) and ZnII-3 (3.0 mM)
indicating that the observed fragments were intermediary pro-
at 50 1C and pH 8.0 (HEPES, 50 mM). Lanes 1 and 12: molecular
weight marker; Lanes 2–9: with ZnII-3; Lanes 10–11: without ZnII-3
(n.c. stands for no complex); Lanes 2, 10, 11: the reaction mixture;
Lanes 3–9: precipitates from the reaction mixture.
ducts and further hydrolysis proceeded during the reaction. BSA
contains 59 lysine residues within total 583 amino acid residues
(Fig. S5(a)w).11 If the ZnII complex hydrolyzes the protein by
binding to the NH2 group of the protein, i.e. the N-terminus and
59 lysine residues, the reaction will yield a large number of small
molecular weight fragments. The above observations on the
SDS-PAGE analysis are consistent with this considerations.z
In order to clarify the role of the lysine residues on the
decomposition of a protein with ZnII-3, porcine pancreatic
elastase seemed to be a more suitable substrate, because this
protein contains only three lysine residues within the total of
240 amino acid residues (Fig. S5(b)w).12 Porcine pancreatic
elastase was found to be rapidly decomposed under pH 9.0,
10.0 or 11.0 even in the absence of ZnII compounds, indicating
that elastase hydrolyzes itself under these conditions. Thus, we
have employed pH 8.0 as the hydrolysis experiment condition
for this protein with a ZnII complex.
Porcine pancreatic elastase (62.4 mM) and the ZnII complex
(3.0 mM) in buffer (pH 8.0, HEPES 50 mM) were heated at
50 1C. The SDS-PAGE analysis of the supernatant of the
mixture showed that decomposition of elastase was considerably
promoted by ZnII-3 (Fig. S7w): the half life was ca. 1 h.y
However, only trace amounts of fragment bands were detected.
Because the reaction mixture with ZnII-3 gave precipitates, the
precipitates were separated from the supernatant by centrifuga-
tion and then treated with ethylenediaminetetraacetate, SDS,
2-mercaptoethanol and glycerin. The resulting solutions were
analyzed by SDS-PAGE. In order to analyze the product at
each reaction time, the reaction mixture was separately placed in
each reaction vessel, reacted at 50 1C for each reaction time, then
each was treated as described above. As shown in Fig. 1,
fragment bands appeared, whose molecular weights were ca.
10 and 5 kDa. The observed fragments in the precipitates were
essentially identical throughout the 0.5 h–24 h reaction period.
The fragments were further analyzed by MALDI-TOF/MS.
The results were shown in Fig. 2. In contrast to elastase which
gave a peak at 25 940, the precipitates formed during the
reaction of elastase and ZnII-3 gave fragment peaks: the
dominant peaks were 8870, 9090 and 5880. The SDS-PAGE
analysis and MALDI-TOF/MS analysis were very consistent
with each other and these results indicate that the scission by
ZnII-3 is selective. The role of the lysine residues in the ZnII-3
assisted scission of a protein was further evidenced by experi-
ments using human elastase instead of porcine pancreatic
elastase under similar conditions (Fig. S8w). Human elastase
involves no lysine residue within the total 218 amino acid
residues (Fig. S5(b)w).13 In the reaction mixture of human
elastase with ZnII-3, no noteworthy protein fragment corres-
ponding to those due to the reaction of porcine pancreatic
Fig. 2 MALDI-TOF/MS analysis: (a) porcine pancreatic elastase; (b)
precipitates from the reaction mixture of porcine pancreatic elastase
(62.4 mM) and ZnII-3 (3.0 mM) at pH 8.0 and 50 1C.
ꢀc
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This finding will open an avenue for using ZnII as a catalytic
center in an artificial peptidase. Furthermore, the present ZnII
complex system will be useful in elucidating the important
factor for the metal ion catalysts in enzymes.
Notes and references
z MALDI-TOF/MS of the mixture of BSA and ZnII-3 (1 : 100) showed
broad peaks in the m/z range 69 000–74 000, in contrast to a sharp
peak of BSA at 66 770 (Fig. S11w). The observations strongly indicate
the binding of 3 to BSA (The number of the bound 3 molecule is
estimated to be ca. 7–24 per one BSA molecule).
y Observed rate constants kobs with or without ZnII-3 determined from
the decrease in the elastase band were 0.63 hꢁ1 and 0.089 hꢁ1 at pH 8.0
(HEPES 50 mM) and 50 1C, respectively.
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Fig. 3 The steric structure of porcine pancreatic elastase (protein
data bank (PDB) ID: 1B0E). Possible scission sites are indicated by
arrows. For details, see ESI.w
elastase with ZnII-3 was observed. Furthermore, the decompo-
sition in the presence of ZnII-3 was rather slower than the
control experiment without ZnII-3. The results strongly sup-
port the important role of the lysine residues in the protein for
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ture of porcine pancreatic elastase indicated that scissions at
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ca. 8790, 8950, 9060 and 5880 Da fragments (Fig. 3 and S9w).
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then promotes hydrolysis of sterically accessible peptide bonds.
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hydrolysis (Fig. S10w).
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In conclusion, a ZnII complex having an aldehyde group,
ZnII-3, was found to cleave a protein backbone under mild
conditions. The stable binding of the ZnII complex to
the protein through a covalent bond appears to be essential.
The results suggest that ZnII can be very active toward peptide
hydrolysis when it strongly binds to the substrate polypeptide.
ꢀc
This journal is The Royal Society of Chemistry 2009
1546 | Chem. Commun., 2009, 1544–1546