Synthesis of hydroxymethylglutathione from glutathione and L-serine catalyzed by carboxypeptidase Y.

Article abstract of DOI:10.1271/bbb.67.434

Hydroxymethylglutathione (gamma-L-glutamyl-L-cysteinyl-L-serine; hmGSH) occurs in many species belonging to the family Gramineae, but the biosynthetic pathway for hmGSH has not been identified. We found that carboxypeptidase Y (CPY), but not carboxypeptidase A, catalyzed hmGSH synthesis from glutathione and L-serine in vitro at acidic pH. CPY also catalyzed methylglutathione synthesis from glutathione and L-alanine. These findings suggested that a carboxypeptidase-like enzyme may be involved in hmGSH synthesis in vivo.

Full text of DOI:10.1271/bbb.67.434

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Synthesis of Hydroxymethylglutathione
from Glutathione and L-Serine Catalyzed by
Carboxypeptidase Y
Ryosuke OKUMURAa, Yukio KOIZUMIa & Jiro SEKIYAa
a Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto
UniversityKyoto 606-8502, Japan
Published online: 22 May 2014.
To cite this article: Ryosuke OKUMURA, Yukio KOIZUMI & Jiro SEKIYA (2003) Synthesis of Hydroxymethylglutathione from
Glutathione and L-Serine Catalyzed by Carboxypeptidase Y, Bioscience, Biotechnology, and Biochemistry, 67:2, 434-437,
DOI: 10.1271/bbb.67.434
To link to this article: http://dx.doi.org/10.1271/bbb.67.434
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Biosci. Biotechnol. Biochem., 67 (2), 434–437, 2003
Note
Synthesis of Hydroxymethylglutathione from Glutathione
and
L
-Serine Catalyzed by Carboxypeptidase Y
†
Ryosuke OKUMURA, Yukio KOIZUMI, and Jiro SEKIYA
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Kyoto 606-8502, Japan
Receivd August 29, 2002; Accepted October 28, 2002
Hydroxymethylglutathione (
-serine; hmGSH) occurs in many species belonging
g
-
L
-glutamyl- -cysteinyl-
L
catalyzed by CPY.
L
hmGSH for use as the standard was synthesized by
the solid-phase method from 9-‰uorenylmethoxycar-
to the family Gramineae, but the biosynthetic pathway
for hmGSH has not been identiˆed. We found that
carboxypeptidase Y (CPY), but not carboxypeptidase
A, catalyzed hmGSH synthesis from glutathione and
serine in vitro at acidic pH. CPY also catalyzed methyl-
glutathione synthesis from glutathione and -alanine.
These ˆndings suggested that a carboxypeptidase-like
enzyme may be involved in hmGSH synthesis in vivo
bonyl (Fmoc)- -glutamate-
L
a
-
tert-butylester, Fmoc-
tert-butyl-
-serine-Alko resin. The synthesized hmGSH was
S-
p
-methoxybenzyl- -cysteine, and Fmoc-O-
L
9)
L
-
L
examined by LC-MS (Shimadzu LCMS-2010A); the
L
observed m z (338) agreed with that of the protonat-
W
ed molecular ion ([M+H]+) of hmGSH. hGSH was
.
prepared by the solid-phase method also.10)
g-GluCys
was a product of Kohjin Co., Ltd. (Tokyo, Japan).
The standard reaction mixture (ˆnal volume
Key words: aminolysis;
carboxypeptidase
Y;
glutathione; hydroxymethylglutathione;
methylglutathione
100
m
l) contained 40 m
M
2-morpholinoethanesulfon-
ic acid (MES)-NaOH buŠer (pH 5.5), 2 m EDTA,
M
0.2 m
and 3
M
dithiothreitol, 1 m
M
GSH, 50 m
M
L
-serine,
Hydroxymethylglutathione
(
g
-
L
-glutamyl-
L
-
m
g of CPY (Oriental Yeast Co., Ltd., Tokyo).
cysteinyl- -serine; hmGSH) occurs in many species
L
After incubation of the reaction mixture for 30 min
belonging to the family Gramineae, including rice,
at 30
15 l of 15 m
Derivatization with monobromobimane was stopped
by the addition of 310 l of 10 acetic acid after
15 min of incubation at 30 C. A portion (20 l) of
9
C, 220
m
l of 200 m
M
Tris-HCl (pH 8.0) and
wheat, and barley.1,2) The glycine residue in GSH is
m
M
monobromobimane were added.11)
replaced with an
thought earlier that hmGSH is synthesized from
glutamyl- -cysteine ( -GluCys) and -serine via
L
-serine residue in hmGSH. It was
g
-
L
-
m
z
L
g
L
a
9
m
reaction similar to that for GSH synthesis, but there
has been no evidence of the involvement of a GSH
synthetase-like enzyme.
the derivatized sample was injected into an HPLC
equipped with a Wakopak Handy ODS column
(4.6
acid, 10:90:0.25 by volume, pH 3.9) and solution B
(methanol water acetic acid, 90:10:0.25 by volume,
×
150 mm). Solution A (methanol water acetic
W
W
Carboxypeptidase Y (CPY, EC 3.4.16.5) is a yeast
vacuolar carboxypeptidase that catalyzes the removal
of the C-terminal amino acid from peptides.3) In
addition, CPY has aminolytic activity by replacing
C-terminal amino acid of the peptide with a free
amino acid.4,5) Therefore, CPY seems to participate
in the turnover and proteolytic processing of pro-
teins.6) Both hydrolysis and aminolysis involve en-
zyme-peptide complex formation and deacylation of
the complex by such nucleophiles as water and free
amino acids, respectively. Phytochelatins also are
synthesized from GSH by a carboxypeptidase-
catalyzed reaction.7,8) We have attempted to synthe-
size hmGSH with CPY. In this communication, we
W
W
pH 3.9) were used as the standard mobile phase at a
ratio of 98:2, and the ‰ow rate was 1 ml min.
Fluorescence of the monobromobimane derivatives
was detected at 480 nm with excitation at 380 nm.
Standard curves were made with authentic
and hmGSH.
W
g-GlyCys
When GSH was incubated with a high concentra-
tion of -serine in the presence of CPY, two new
L
peaks, 1 and 2, appeared (Fig. 1). The retention times
of peaks 1 and 2 were consistent with those of the
derivatives of authentic
monobromobimane, respectively. In addition, the
retention times of the peaks predicted to be -GluCys
g-GluCys and hmGSH with
describe hmGSH synthesis from GSH and
L
-serine
g
†
To whom correspondence should be addressed. Tel: +81-75-753-6109; Fax: +81-75-753-6128; E-mail: jiro
＠
kais.kyoto-u.ac.jp
Abbreviations
:
CPY, carboxypeptidase Y; -GluCys, -glutamyl- -cysteine; Fmoc, 9-‰uorenylmethoxycarbonyl-; hGSH,
g
g-
L
L
homoglutathione; hmGSH, hydroxymethylglutathione; MES, 2-morpholinoethanesulfonic acid; mGSH, methylglutathione; PMSF,
phenylmethylsulfonyl ‰uoride

Hydroxymethylglutathione Synthesis
435
Fig. 2. Changes with Time in Formation of
hmGSH Catalyzed by CPY.
The standard reaction mixture was incubated for the times in-
dicated, and -GluCys and hmGSH were measured as described
in the text. -GluCys ( ); hmGSH ( ).
g-GluCys and
g
g


Fig. 1. HPLC Proˆles of Reaction Products Obtained with
Active (A) and Heat-denatured (B) CPY.
The standard reaction mixture described in the text was used
for A and the active CPY was replaced with the heat-denatured
CPY for B. Peak 1, g-GluCys; peak 2, hmGSH; peak 3, GSH.
Fig. 3. EŠects of pH on the Formation of g-GluCys and hmGSH
Catalyzed by CPY.
The following buŠers were used instead of 40 m
NaOH buŠer (pH 5.5) in the standard reaction mixture; 40 m
acetate buŠer (pH 4.0), 40 m MES-NaOH buŠer (pH 5–6), 40
HEPES-NaOH buŠer (pH 7.0) and 40 m Tris-HCl buŠer
(pH 8–9). -GluCys ( ); hmGSH ( ).
M MES-
and hmGSH derivatives also were consistent with
those of authentic -GluCys and hmGSH derivatives,
respectively, when a gradient of 5–20 methanol in
20 m Na2HPO4 adjusted to pH 6.0 with acetic acid
was used as the mobile phase.1) However, the peaks
corresponding to -GluCys and hmGSH derivatives
did not appear when heat-denatured CPY was used,
or when no CPY was used (not shown). When
M
g
M
m
M
M
z
g


M
g
pH 7.0 (Fig. 3). The amounts of hmGSH formed
were almost constant at pH 5.5 or below. The
amounts of g-GluCys increased with decreasing pH.
D
-
serine was the substrate, aminolysis catalyzed by
CPY did not proceed. These ˆndings indicate that
hmGSH was synthesized stereospeciˆcally from GSH
In general, CPY-catalyzed aminolysis proceeds at
basic pH, but the optimum pH for the hydrolysis is
5.5–6.5.4,5,12) However, our ˆndings on hmGSH syn-
thesis diŠer from those of other aminolytic reactions
reported. Perhaps the amino group of the amino acid
that would otherwise act as the nucleophile is proto-
nated at acidic pH. A protonated amino group can
not act as the nucleophile needed to attack the acyl-
enzyme intermediate, so aminolysis seldom occurs at
and
L
-serine by the enzyme reaction catalyzed by
CPY. In contrast, carboxypeptidase A (EC 3.4.17.1)
from bovine pancreas did not have any activity caus-
ing hmGSH synthesis from GSH and
ic or basic pH, although it catalyzed the hydrolysis of
GSH into -GluCys.
L
-serine at acid-
g
The amount of hmGSH increased linearly with
the reaction time for 1 h, followed by a slow increase
up to 4 h (Fig. 2). After this time, the amount of
hmGSH formed became constant. In contrast, the
acidic pH. When the enzyme-substrate (CPY-g-Glu-
Cys) complex forms, however, the three-dimensional
structure of CPY may be changed, resulting in
aminolysis regardless of the scarcity of a nonionized
amino group of the amino acid as a nucleophile.
amount of
g
-GluCys continued to increase linearly
-GluCys was ap-
for 5 h. The ratio of hmGSH to
g
proximately 1:5 under the reaction conditions used.
More hmGSH was synthesized at acidic than at
basic pH; a small amount of hmGSH was detected at
Higher
mixture led to more hmGSH synthesized (Fig. 4), but
the amount of -GluCys formed was roughly con-
L
-serine concentrations in the reaction
g

436
R. OKUMURA et al.
Table 1. Retention Times and Fluorescence Intensities of
Monobromobimane Derivatives of GSH and Related Compounds
Compound
-GluCys
hmGSH
GSH
mGSH
hGSH
Relative retention time
Relative intensity
g
0.77
0.86
1.00
1.25
1.62
1.09
0.93
1.00
a)
—
0.95
a)
not determined.
hmGSH, all of which occur in the plant kingdom.
Gramineous plants such as barley, wheat, and rice
have multiple forms of serine-type carboxypepti-
dase.13–16) When we measured the enzyme activity
toward hmGSH synthesis using a crude extract of
Fig. 4. EŠects of -Serine Concentration on Formation of g-Glu-
L
Cys and hmGSH Catalyzed by CPY.
L
-Serine concentrations in the reaction mixture were in the
range of 0 to 400 m . g-GluCys ( ); hmGSH ( ).
M
 
rice leaves, hmGSH was formed from GSH and -
L
stant in the range of 0–100 mM L-serine and decreased
at higher concentrations of -serine. At -serine
concentrations higher than 200 m , the amount
of hmGSH formed exceeded that of -GluCys.
, which was
serine, probably by an aminolysis reaction of carbox-
ypeptidase (not shown). These ˆndings suggested
that carboxypeptidase or a carboxypeptidase-like
L
L
M
g
enzyme may be involved in hmGSH synthesis in vivo.
However, the
K
m for
L
-serine was 330 m
fairly high value. This high Km may be a reason why
M
Acknowledgments
yeast cells do not synthesize hmGSH.
When CPY (50
1 m
30 min at 30
m
g in 100
m
l) was incubated with
We thank Dr. M. Miyashita for hmGSH synthesis
by the solid-phase method and Dr. T. Ishihara for
LC-MS measurement.
M
phenylmethylsulfonyl ‰uoride (PMSF) for
C and then excess PMSF was removed
9
by gel ˆltration with Sephadex G-25, the enzyme
activity of CPY was completely inhibited. This
ˆnding indicates that the serine residue in CPY was
essential for aminolysis. CPY catalyzed the synthesis
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