Metal-ion-assisted hydrolysis of dipeptides involving a serine residue in a neutral aqueous solution.

Article abstract of DOI:10.1039/b209565c

Dipeptides having a serine residue at the C-terminus, X-Ser, where X is an appropriate amino acid residue, were efficiently hydrolyzed in the presence of ZnCl2 at pH 7.0. The rapid hydrolysis of X-Ser is due to an autocatalysis of the hydroxy group in the serine residue, and is found to be accelerated by a metal ion, in particular by ZnCl2. Roles of the metal ion in the hydrolysis of peptides involving a serine residue, in relation to the recently reported protein cleavages, are discussed.

Full text of DOI:10.1039/b209565c

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Metal-ion-assisted hydrolysis of dipeptides involving a serine
residue in a neutral aqueous solution†
a
b
b
b
b
Morio Yashiro,* Yoko Sonobe, Ai Yamamura, Tohru Takarada, Makoto Komiyama
c
and Yuki Fujii
a
Department of Applied Chemistry, Faculty of Engineering, Tokyo Institute of Polytechnics,
1583 Iiyama, Atsugi, Kanagawa 243-0297, Japan. E-mail: yashiro@chem.t-kougei.ac.jp;
Fax: ϩ81 (0)46-242-9554; Tel: ϩ81 (0)46-242-9554
b
c
Department of Chemistry and Biotechnology, Graduate School of Engineering,
The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
Department of Chemistry, Faculty of Science, Ibaraki University, Bunkyo 2-1-1,
Mito 310-8512, Japan
Received 2nd October 2002, Accepted 20th December 2002
First published as an Advance Article on the web 23rd January 2003
Dipeptides having a serine residue at the C-terminus, X-Ser, where X is an appropriate amino acid residue, were
efficiently hydrolyzed in the presence of ZnCl at pH 7.0. The rapid hydrolysis of X-Ser is due to an autocatalysis
2
of the hydroxy group in the serine residue, and is found to be accelerated by a metal ion, in particular by ZnCl2.
Roles of the metal ion in the hydrolysis of peptides involving a serine residue, in relation to the recently reported
protein cleavages, are discussed.
Table 1 Results of the hydrolysis of dipeptides in the presence of
Introduction
a
ZnCl at pH 7.0 (0.1 M HEPES), 70 ЊC and 24 h
2
The non-enzymatic hydrolysis of peptides has been attracting
1–7
much attention. Recently, the peptide hydrolyses promoted
Entry
Dipeptide
Conv (%)
2ϩ 2
2ϩ 3
2ϩ 3a,b
2ϩ/3ϩ 4
2ϩ 5
4ϩ 6
by Cu , Pd , Pt , Fe
, Ni
and Ce
under either
near neutral or acidic conditions have been reported. The
studies indicate that catalysts involving these transition metal
ions are promising as non-enzymatic cleavage reagents under
1
2
3
4
5
6
7
8
9
0
1
2
3
Gly-Ser
Gly-Ser
Gly-Ser
Ser-Gly
Phe-Ser
Leu-Ser
Ile-Ser
Pro-Ser
His-Ser
Val-Ser
Gly-Thr
Thr-Gly
Gly-Asp
Gly-Asn
Gly-Gly
Gly-Ala
83
b
22c
6
4
1
near physiological conditions. The site specific cleavages of
91
81
10
80
49
14
35
2
5
2
6
6
2ϩ 3d
2ϩ 2d
2ϩ 5
proteins in the presence of Pd , Cu
reported.
and Ni
have been
It has been known that proteins involving a serine or a
threonine residue undergo hydrolysis via an N O acyl
rearrangement under non-physiological extreme acidic or basic
1
1
1
1
8,9
conditions. The reaction proceeds by an intramolecular
attack of the hydroxy group in the serine or the threonine
residue on the carbonyl carbon in the preceding amide bond to
form an ester intermediate. Under the conditions where the
rapid hydrolysis of the ester intermediate occurs, the reaction
14
15
1
6
8
a
b
c
results in the peptide hydrolysis by a two-step-reaction.
[dipeptide] = 10 mM, [ZnCl ] = 10 mM. At 50 ЊC. Without ZnCl2.
2
Recently, the N O acyl rearrangement has been attracting
much attention in relation to important biological events; some
post translational modifications of proteins, such as protein
splicing, autoproteolysis and autoprocessing, have been known
to proceed self-catalytically, and the first step of these reactions
Results and discussion
Hydrolysis of dipeptides with ZnCl2
10
is the N O acyl rearrangement. Many promising applic-
ations of the protein splicing have been demonstrated by using
parts of naturally occurring proteins that can promote protein
The hydrolysis of dipeptides (10 mM) were conducted in the
presence of ZnCl (10 mM) at pH 7.0 and 70 ЊC. The starting
2
10,11
peptide and the resulting amino acids were separated and
splicing.
Thus, finding a way to accelerate reactions involv-
2b,6b,12
determined by HPLC.
With the decrease of the starting
ing the N O acyl rearrangement, and, accordingly, to regulate
them will open an avenue for the site-specific cleavage or the
modification of a protein.
This study deals with the effect of a metal ion on the
hydrolysis of peptides involving a serine residue. We have
focused on the hydrolysis of dipeptides, because the coordin-
ation behaviour of the metal ion and the functional groups
in the peptide are relatively simple compared with longer
peptides.
dipeptide, corresponding α-amino acids involved in the starting
peptide were formed. No other amino acid or peptide was
detected. The mass balance before and after the reaction was
sufficiently good (at least 90%), indicating that the hydrolysis of
the peptide bond was the main reaction under the present
experimental conditions. The conversions estimated from the
formation of amino acids are summarized in Table 1.
Obviously, the conversion was largely dependent on the
sequence of dipeptides. The conversion after the 24 h reaction
at pH 7.0 and 70 ЊC ranged from 2% (Thr-Gly, Gly-Asn) to 91%
†
Electronic supplementary information (ESI) available: Kinetic studies
(
Phe-Ser). Among dipeptides examined, Gly-Ser, Phe-Ser,
involving pH and concentration profiles of the rate constant. See http://
www.rsc.org/suppdata/ob/b2/b209565c/
Leu-Ser, Pro-Ser and His-Ser were the most readily hydrolyzed.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 2 9 – 6 3 2
629

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Ile-Ser, Val-Ser and Gly-Thr were moderately active for the
hydrolysis. All dipeptides that were readily hydrolyzed have
a serine residue at the C-terminus, indicating the important role
of the Ser hydroxy group on the peptide hydrolysis as an intra-
molecular nucleophile, as expected. The interpretation that the
hydrolysis of X-Ser is due to the N O acyl rearrangement
is strongly supported by the marked difference between the
conversion of Gly-Ser (83%) and that of Ser-Gly (4%). The
intramolecular attack of the Ser hydroxy group on the amide
carbonyl carbon is possible in Gly-Ser, while it is impossible in
Ser-Gly, because in the former case the intramolecular attack of
the Ser hydroxy group results in a five-membered ring transition
state, while in the latter case the intramolecular attack should
form an unfavorable four-membered ring transition state
Fig. 2 Two types of coordination of a metal ion to a dipeptide. (a) The
amide carbonyl group is activated for the hydrolysis. (b) The amide
carbonyl group is inactivated for the hydrolysis.
(
Fig. 1). The corresponding ester intermediate was not detected
in any reaction of X-Ser, but only corresponding amino acids
were detected.
Fig. 3 A comparison of the sequence dependence of the metal assisted
hydrolysis of dipeptides at 70 ЊC, 24 h and pH 7.0 (0.1 M HEPES).
[Dipeptide] = 10 mM, [metal chloride] = 10 mM.
Fig. 1 A comparison of Gly-Ser and Ser-Gly.
Sequence dependence of the metal-ion-assisted hydrolysis of the
dipeptide
Hydrolysis of Gly-Ser with various metal salts
Fig. 3 shows the comparison of the hydrolyses of three
dipeptides, Gly-Gly, Ser-Gly and Gly-Ser, in the presence of
Effects of various metal ions on the hydrolysis of Gly-Ser were
examined at pH 7.0 and 70 ЊC. Results are summarized in
Table 2. All transition metal salts examined, except CuCl2,
showed considerable effects. ZnCl2 was the most effective
among metal salts examined.
ZnCl , NiCl , CdCl and EuCl . When ZnCl , NiCl and CdCl
2
2
2
3
2
2
2
were used, Gly-Ser was hydrolyzed selectively. In the presence
of EuCl , however, not only Gly-Ser, but also Gly-Gly and
3
Ser-Gly were hydrolyzed considerably. We have previously
reported that lanthanide salts promote the hydrolysis of
The low conversion with CuCl is reasonably explained as the
2
6
2ϩ
dipeptides very efficiently. The present results indicate that,
following: Cu is known to bind to a dipeptide through NH2
N-terminal), NH (amide) and COO (C-terminal) groups to
Ϫ
compared with the strong catalytic effect of EuCl3 on the
peptide hydrolysis due to an external nucleophile, the effect of
the Ser hydroxy group is relatively small.
Thus, a highly preferential hydrolysis of Gly-Ser over
Gly-Gly and Ser-Gly was achieved in the presence of ZnCl2,
(
form a stable tridentate chelate (Fig. 2b). Because the amide
proton in such a chelate readily dissociates under neutral or
weakly basic conditions, the dipeptide is inactive for the
2b,13
hydrolysis under such conditions.
When the formation of
NiCl and CdCl .
the tridentate chelate of a dipeptide is not favored upon using
2
2
2
b
2c
[
CuCl (tach)] or [CuCl ([9]aneN )] (tach = cis,cis-1,3,5-
2
2
3
triaminocyclohexane, [9]aneN = 1,4,7-triazacyclononane), the
Rate constants for the hydrolysis of X-Ser
3
2ϩ
dipeptide hydrolysis is efficiently promoted by the Cu com-
2b,c
In order to study the hydrolysis of dipeptides due to the
combination of the Ser hydroxy group and ZnCl in more
detail, kinetic studies of the reactions of X-Ser were conducted
plex under nearly neutral conditions. Contrary to this, in the
2ϩ
2ϩ
2
case of Zn and other transition metal ions except Cu , the
tridentate chelate of a dipeptide would be formed only margin-
ally, and a high hydrolysis activity would be observed (Fig. 2a).
with or without ZnCl at pH 7.0 (0.1 M HEPES) and 50 ЊC. The
2
reactions were followed by the formation of the corresponding
amino acids, i.e. X and Ser. The pseudo-first-order rate con-
stants are summarized in Table 3.
The rate constant was significantly dependent on X. When
X of the dipeptide was Ala, Leu or Phe, the rate was greater
Table 2 Effects of metal ions on the hydrolysis of Gly-Ser at pH 7.0
a
(
0.1 M HEPES) and 70 ЊC
b
Entry
Metal chloride
Conv (%)
Table 3 Pseudo-first-order rate constants for the hydrolysis of X-Ser
at pH 7.0 (0.1 M HEPES) and 50 ЊC
1
2
3
4
5
6
7
8
9
0
1
2
3
none
MgCl2
6
8
6
52
6
83
43
28
49
66
58
30
13
CaCl2
Ϫ3 Ϫ1
kobs/10
h
NiCl2
CuCl2
a
With ZnCl2 b
Dipeptide
Without ZnCl2
ZnCl2
CdCl2
LaCl3
CeCl3
PrCl3
EuCl3
ErCl3
LuCl3
Ala-Ser
Phe-Ser
Leu-Ser
Gly-Ser
Val-Ser
Ile-Ser
24 ± 4
32 ± 8
22 ± 3
12 ± 2
2.0 ± 0.2
1.22 ± 0.04
2.0 ± 0.2
1.4 ± 0.4
1.6 ± 0.4
0.58 ± 0.05
0.26 ± 0.04
0.26 ± 0.05
1
1
1
1
a
b
a
b
[
Gly-Ser] = 10 mM, [metal chloride] = 10 mM. After 24 h reaction.
[Dipeptide] = 10 mM. [ZnCl ] = 10 mM.
2
6
30
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 2 9 – 6 3 2

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than that of Gly-Ser. In contrast, when X was Val or Ile, the
rate was smaller than that of Gly-Ser.
In the case of Val and Ile, the side chain has a branched
structure at the β-position, while the side chain of Ala, Leu or
Phe has no branched structure at the β-position (Fig. 4). The
Scheme 1
would be accelerated. Recently, highly site-specific cleavage of
2ϩ 5
2ϩ 3d
2ϩ 2d
proteins assisted by a metal ion, i.e. Pd , Cu , or Ni ,
have been reported. Since these reactions resulted in X-Ser
or X-Thr bond cleavage, their reaction mechanisms could be
1c
similar to those in the present reactions.
2ϩ
The reaction mechanism for Zn -assisted peptide hydrolysis
could be better illustrated on the basis of kinetic studies involv-
ing the pH profile or the concentration profile of the rate
constant (see Electronic supplementary information†). The
kinetic data in the pH region above pH 7 are of special interest
with respect to discussing the nature of the nucleophile. How-
ever, such experiments could not be conducted because of the
solubility of ZnCl ; insoluble Zn compounds were formed in
2
the pH region higher than 7. Instead, kinetic studies using a
2ϩ
Zn complex with an appropriate ligand would be useful.
Fig. 4 Structures of dipeptides whose hydrolysis rates were greater
than Gly-Ser (a), and those whose hydrolysis rates were smaller than
Gly-Ser (b).
Conclusion
A rapid hydrolysis of a dipeptide under neutral conditions was
achieved by the combination of an internal hydroxy group and
alkyl side chain with the β-branched structure would cause a
steric hindrance therefore inhibiting a nucleophilic attack,
resulting in a relatively slow peptide hydrolysis. In contrast,
quite interestingly, when the alkyl side chain in X does not have
a β-branched structure, the hydrolysis is accelerated. Such an
effect of X is almost independent of Zn , since the rate
constants for the reactions both with and without ZnCl2
showed similar tendencies.
ZnCl . The reaction of X-Ser proceeds self-catalytically, and is
2
2ϩ
accelerated by a transition metal ion, in particular by Zn .
It has been suggested that the protein degradation via the
2
ϩ
2ϩ
N
O acyl rearrangement in vivo can be assisted by Cu or
2ϩ 1c 2ϩ
Ni . The present results suggest that Zn could also acceler-
ate the self-catalytic reactions of proteins via the N O acyl
10
rearrangement.
Role of the metal ion
Experimental
The present results indicate that the metal ion, in addition to
the hydroxy group within the peptide, plays an important role
Materials
2ϩ
in the rapid hydrolysis of a dipeptide. In the Cu -complex-
2ϩ
Dipeptides were purchased from Sigma-Aldrich Co. All other
reagents were purchased from commercial chemical sources
and used as received.
assisted hydrolysis of Gly-Ser, the coordination of Cu to the
carbonyl oxygen of the peptide was found to be an essential
2a,b
factor. Moreover, it has been known that bovine lens leucine
aminopeptidase catalyzes the peptide hydrolysis by using two
2
ϩ
2ϩ
Zn ions in the catalytic center. One of roles of Zn is
suggested to be the ability to polarize the carbonyl group by
Hydrolysis of dipeptides in the presence of metal salts. In a
typical experiment, to a 10 ml aqueous buffer (pH 7.0, 100 mM
HEPES) were added Gly-Ser (0.1 mmol) and ZnCl (0.1 mmol).
The pH of the resulting solution was adjusted to 7.0 at rt
(20 ЊC) with 0.1 M NaOH, and heated at 70 ЊC for 24 h. The
reaction temperature was controlled using a Lauda thermostat
M3T. The reaction mixture was analysed by HPLC [Column,
TOSOH TSKgel Aminopak; eluent, citrate buffer (67 mM, pH
14
coordination to facilitate the attack of the nucleophile. Thus,
a possible role of Zn in the present reaction is to coordinate to
the carbonyl oxygen and to assist the attack of the nucleophile.
2
2ϩ
2ϩ
The coordination of Zn with the dipeptide through NH and
2
13
C᎐O is the most probable (Fig. 2a). Such coordination of
᎐
2ϩ
Zn with a dipeptide has been confirmed by X-ray crystallo-
15
Ϫ1
graphy. The overall reaction scheme is shown in Scheme 1.
3.41 or 4.25); flow rate, 0.6 ml min ] and was fluorometrically
The coordination of a metal ion to the NH group is import-
detected (ex 345 nm, em 455 nm) after the on-line post-column
2
ant only in assisting coordination to the C᎐O group. Therefore,
in the case of a longer peptide or a protein, when the coordin-
derivatizing reaction with o-phthalaldehyde–2-mercaptoethanol
᎐
2b,6b,12
at 60 ЊC.
The detected species were identified by compar-
ation of a metal ion to the C᎐O group is facilitated by the
ing their elution times with those of standard samples. The
quantification of each detected species was carried out by com-
paring the peak area with that of the corresponding standard
sample.
᎐
assistance of appropriate functional group(s) in the amino acid
residue(s) in the stereochemical vicinity of the target amide
bond, the peptide bond scission via the N O rearrangement
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 2 9 – 6 3 2
631

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Kinetic constant measurement of the hydrolysis of X-Ser in the
Milinkovic, T. N. Parac, M. I. Djuran and N. M. Kostic, J. Chem.
Soc., Dalton Trans., 1997, 2771–2776; (j) G. B. Karet and
N. M. Kostic, Inorg. Chem., 1998, 37, 1021–1027; (k) T. N. Parac and
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presence of ZnCl . In a typical experiment, to 10 ml aqueous
2
buffer (pH 7.0, 100 mM HEPES) were added Gly-Ser (0.1
mmol) and ZnCl (0.1 mmol). The pH of the resulting solution
2
was adjusted to 7.0 (20 ЊC) with 0.1 M NaOH, and heated at
0 ЊC. After appropriate reaction periods, the reaction mixture
3
83–391; (m) X.-H. Chen, L.-G. Zhu, X.-Z. You and N. M. Kostic,
5
J. Biol. Inorg. Chem., 1998, 3, 1–8; (n) T. N. Parac, G. M. Ullmann
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was subjected to the analysis as described above. The reactions
were followed for at least 30 h. Within this time period, all
2b
reactions showed pseudo-first-order kinetics.
4
5
Acknowledgements
1
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The authors thank Prof. Kazuhiko Saigo. This work was
partially supported by a Grant-in-Aid for Scientific Research
no. 12650845) from the Ministry of Education, Culture,
Sports, Science and Technology, Japan.
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O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 2 9 – 6 3 2