Substrate specificity of aminopeptidase from the mid-gut gland of the scallop (Patinopecten yessoensis)

Article abstract of DOI:10.1271/bbb.68.945

An action for various peptides and a kinetic study for ami no acid p-nitroanilides (pNAs) and 4-methylcoumaryl-7-amides (MCAs) were performed with purified aminopeptidase from the mid-gut of the scallop. The enzyme preferred dipeptides having Ala, Met, and Phe in the amino-terminal or the penultimate position from the amino-termini. The catalytic efficiencies, k _cat/K_m values for Ala-pNA and MCA were the highest in the tested substrates, and those for pNA and MCA substrates having Met or Phe were the next highest. The enzyme was found to be a new alanine-specific aminopeptidase.

Full text of DOI:10.1271/bbb.68.945

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Substrate Specificity of Aminopeptidase from
the Mid-gut Gland of the Scallop (Patinopecten
yessoensis)
ab
a
b
c
c
Hironori UMETSU , Mito ARAI , Toshinori OTA , Kaoru ABE , Hidemitsu UCHIZAWA &
b
Kazuo SASAKI
a
Graduate School of Environmental Science, Aomori University
b
Institute of Bioscience and Biotechnology, Aomori University
c
Aomori Industrial Research Center
Published online: 22 May 2014.
To cite this article: Hironori UMETSU, Mito ARAI, Toshinori OTA, Kaoru ABE, Hidemitsu UCHIZAWA & Kazuo SASAKI (2004)
Substrate Specificity of Aminopeptidase from the Mid-gut Gland of the Scallop (Patinopecten yessoensis), Bioscience,
Biotechnology, and Biochemistry, 68:4, 945-947, DOI: 10.1271/bbb.68.945
To link to this article: http://dx.doi.org/10.1271/bbb.68.945
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Biosci. Biotechnol. Biochem., 68 (4), 945–947, 2004
Note
Substrate Specificity of Aminopeptidase from the Mid-gut Gland
of the Scallop (Patinopecten yessoensis)
1
;2;y
1
2
3
Hironori UMETSU,
Mito ARAI, Toshinori OTA, Kaoru ABE,
3
2
Hidemitsu UCHIZAWA, and Kazuo SASAKI
1
Graduate School of Environmental Science, Aomori University, Kobata, Aomori 030-0943, Japan
Institute of Bioscience and Biotechnology, Aomori University, Kobata, Aomori 030-0943, Japan
Aomori Industrial Research Center, Daini-tonyamachi, Aomori 030-0013, Japan
2
3
Received October 30, 2003; Accepted January 24, 2004
An action for various peptides and a kinetic study for
amino acid p-nitroanilides (pNAs) and 4-methylcouma-
ryl-7-amides (MCAs) were performed with purified
aminopeptidase from the mid-gut of the scallop. The
enzyme preferred dipeptides having Ala, Met, and Phe
in the amino-terminal or the penultimate position from
the amino-termini. The catalytic efficiencies, kcat=Km
values for Ala-pNA and MCA were the highest in the
tested substrates, and those for pNA and MCA sub-
strates having Met or Phe were the next highest. The
enzyme was found to be a new alanine-specific amino-
peptidase.
gel electrophoresis and an isoelectric point of 5.2. The
enzyme preferred substrates having Ala or Met as an
amino acid residue from the results of relative hydrol-
ysis rates of amino acid-pNAs and MCAs. The enzyme
appeared to be classifiable as a cytosol alanyl amino-
peptidase (EC3.4.11.14) with respect to its substrate
specificity, localization, and the need for Zn2 for
enzyme activity, but differs from cytosol alanyl amino-
peptidase in molecular weight and sensitivity to pur-
omycin, SH-blocking reagents, and divalent metal ions.
This paper describes an action of scallop amino-
peptidase on various peptide substrates and a kinetic
study for amino acid-pNAs and MCAs.
þ
Key words: scallop; mid-gut gland; aminopeptidase;
substrate specificity; kinetic parameter
Ala-pNA, Leu-pNA, Ala-MCA, Arg-MCA, Leu-
MCA, Lys-MCA, Met-MCA, Phe-MCA, 7-amino-4-
methylcoumarin (AMC), Gly-Phe, (Gly) , and Ala-Ser-
3
Aminopeptidases (ꢀ-aminoacyl-peptides hydrolase,
EC 3.4.11.1-15) have the ability to liberate various
amino acid residues from the amino-termini of peptide
substrates and are classified according to preference for
amino-terminal amino acid of substrates, their location,
sensitivity to inhibitors, and the need for metal, such as
Thr-Thr-Asn-Tyr-Tyr-Gly were purchased from the
Peptide Institute, Inc. (Osaka, Japan). Asp-MCA, Ser-
MCA, Tyr-MCA, His-Ala, Met-Tyr-Phe, Ala-Gl-Gly,
(Ala)4, and Ala-Phe-Ser-Ser-Trp-Gly were purchased
from Bachem Inc. (Bubendorf, Switzerland), and all
other amino acid-pNAs and peptides from Sigma. The
aminopeptidase was purified from the mid-gut gland of
the scallop and assayed using Ala-pNA as a substrate as
1
)
zinc or cobalt, for their enzyme activities. There are
substantial reports concerning substrate specificity, bio-
logical function, and structure of aminopeptidases in
15)
in our previous experiment. One katal was the amount
of enzyme required to liberate 1 mol p-nitroaniline per
2
,3)
prokaryotes and mammals.
ꢀ
Among various aminopeptidases, the alanyl amino-
peptidases (EC 3.4.11.2, 3.4.11.14), which preferentially
liberate amino-terminal amino acids, such as Ala, Met,
Leu, and Tyr of peptides, are widely distributed in
second from Ala-pNA at pH 7.0 and 30 C. The
fluorometric assay was done as in our previous expri-
15)
ment by the use of amino acid-MCAs as substrates.
The substrate specificity of the scallop aminopepti-
dase was examined with various peptide substrates
(Table 1). The purified enzyme from scallop had a
remaining activity of 91% of its original activity in the
4
–9)
mammalian tissues and body fluids,
0)
and in plant
as a membrane or
tissues1 and cyanobacteria,
11)
cytosolic type. These enzymes from mammals are
believed to participate in the metabolism of hormones
ꢀ
absence of substrate after incubation at pH 7.0 and 30 C
12–14)
and neurotransmitters.
for 24 h. The enzyme acted preferentially on Phe-Met,
Ala-Phe, Met-Phe, Ala-Ala, and Ala-Leu, and then
moderately on Met-Ala, Gly-Phe, and Met-Gly. How-
ever, the enzyme scarcely hydrolyzed dipeptides having
His, such as Ala-His or His-Ala, and those having
In a previous paper,15) we demonstrated that an
aminopeptidase, isolated and purified from the mid-gut
gland of the scallop (Patinopecten yessoensis), is an
enzyme with a MW of 61,000 by SDS-polyacrylamide
y
To whom correspondence should be addressed. Fax: +81-17-738-2030; E-mail: umetsu@aomori-u.ac.jp

946
H. UMETSU et al.
Table 1. Relative Hydrolysis Rates of Various Peptides by the
Action of Aminopeptidase from Scallop
Table 2. Kinetic Parameters of Aminopeptidase from Scallop for
Amino Acid-pNAs and MCAs
ꢃ
Relative activity
%)
Km
kcat
kcat=Km
ꢁ1 ꢁ1
mM )
Substrate
Substrate
(sec 1)
ꢁ
(sec
(
(ꢁM)
ꢂ
ꢃ
Phe-Met
Ala-Phe
158
137
100
94
92
89
32
30
12
tr
Ala-pNA
32.2
34.1
80.6
24.9
71.9
29.5
6.24
3.66
0.87
0.68
921
184
46
Met-pNA
Gly-pNA
Pro-pNA
Leu-pNA
Ala-pNA
Met-Phe
35
Ala-Ala
Ala-Leu
9.4
ꢃ
Ala-MCA
Phe-MCA
Met-MCA
Ser-MCA
Arg-MCA
Tyr-MCA
Leu-MCA
Asp-MCA
Lys-MCA
11.1
1.12
8.70
40.4
3.92
2.11
8.33
16.0
16.7
9.49
0.706
3.84
854
628
441
377
173
130
Met-Ala
Gly-Phe
Met-Gly
Ala-His
15.1
0.680
0.274
0.269
0.108
0.049
His-Ala
Ala-Pro
Met-Pro
tr
tr
32.3
tr
6.8
2.9
Ala-Ala-Ala
Met-Ala-Ser
Ala-Gly-Gly
Gly-Gly-Gly
Met-Tyr-Phe
Ala-Phe-Tyr-Glu
Ala-Ala-Ala-Ala
Ala-Phe-Ser-Ser-Trp-Gly
Ala-Ser-Thr-Thr-Asn-Tyr-Tyr-Gly
32
18
14
8
ꢃ
Data from reference (15).
The enzyme activities for amino acid-pNAs and MCAs were measured in
ꢀ
tr
MacIlvaine buffer (pH 7.0) at 30 C. Enzyme concentration was estimated
1
%
20
tr
from the absorbance at 280 nm by the extinction coefficient, A
value of
cm
1
23.0.15) Each experiment was done at five different substrate concentrations.
The values of Km and kcat were determined graphically from Lineweaver-
Burk plots.
0
0
ꢃ
The rate of hydrolysis of Ala-pNA=100. tr., trace.
The enzyme (1 nkat/ml) and peptide (250 nmol/ml) in 100 mM ammonium
ꢀ
hydrogen carbonate (pH 7.0) were incubated at 30 C for 24 h and the
liberated amino acids were analyzed by an amino acid analyzer.
15)
for Ala-pNA and Ala-MCA
ꢁ1
were 921 and 854
mM , respectively. These values were highest in
ꢁ1
s
ꢂ
the pNA and MCA substrates. In the pNA substrates, the
kcat=Km value for Met-pNA was second highest and
those for the substrates having Gly, Pro, and Leu
decreased in that order. In the MCA substrates, the
enzyme had second highest k =K values of 628 and
imidopeptide bond, such as Ala-Pro or Met-Pro. Among
tripeptide substrates, (Ala) was the most rapidly hydro-
3
lyzed. The hydrolysis rates of Met-Ala-Ser, Ala-Gly-
Gly, and (Gly)3 slowed in that order, and Met-Tyr-Phe
was scarcely attacked by the enzyme. Among peptides
longer than tripeptide, Ala-Phe-Tyr-Glu was hydrolyzed
with a relative hydrolysis of 20% compared to Ala-pNA,
whereas the other peptides tested were scarcely hydro-
lyzed. These findings show that the enzyme prefers
substrates having Ala, Met, and Phe in the amino-
terminal or the penultimate position from the amino-
termini. Compared with the actions for Ala-Ala, (Ala)3,
and (Ala)4, the hydrolysis rate of Ala-Ala was faster and
that of (Ala)3 slower. (Ala)4 was not hydrolyzed by the
enzyme. The enzyme strongly cleaved Ala-Phe, whereas
Ala-Phe-Ser-Ser-Trp-Gly were scarcely attacked. These
results show that the enzyme hydrolyzes most rapidly
dipeptide and tirpeptides slowly, and acts with difficulty
on peptides longer than tripeptide.
cat
m
ꢁ
1
ꢁ1
441 s
ꢂ
mM for Phe- and Met-MCA, respectively.
When Ser, Arg, Tyr, Leu, Asp, and Lys were amino acid
residues in the MCA substrates, the kcat=Km values
decreased in that order. The kcat=Km values for various
pNA and MCA substrates were of the same levels as
those for MCA substrates in alanine aminopeptidase
9
)
from guinea-pig brain, for peptide substrates in rat-
8
)
liver cytosol, and for pNA substrates and dipeptides in
Aeromonas caviae aminopeptidase.
16)
The results of the action for various peptides and the
kinetic study for pNA and MCA substrates showed that
scallop aminopeptidase is specific for alanine in the
amino-termini of substrates and resembles mammal
4
–6,8,9)
cytosol alanyl aminopeptidases
with respect to
15)
substrate specificity. Our previous paper demonstrat-
ed, however, that the enzyme differs from mammal
cytosol alanyl aminopeptidase in molecular weight and
sensitivity to puromycin, SH-blocking reagents, and
divalent metal ions. Judging by these overall results, the
enzyme from the mid-gut gland of the scallop may be
classified into a new category different from cytosol
alanyl aminopeptidase. Further investigation concerning
primary or higher order structure will be required to
verify the category of the scallop enzyme.
Table 2 shows the kinetic parameters of the scallop
aminopeptidase for amino acid-pNAs and MCAs. The
kcat and Km values were affected by amino acid residues
of the substrates. In the pNA substrates, the values of Km
were in the range of 24.9–80.6 ꢁM, and those of kcat
ꢁ1
varied in the range of 0.68–29.5 sec . In the MCA
substrates, the values of K and k varied dramatically
m
cat
ꢁ1
in the range of 1.12–40.0 ꢁM and 0.049–15.1 sec
,
respectively. The catalytic efficiencies, kcat=Km values

Aminopeptidase from Mid-gut Gland of Scallop
peptides. Int. J. Biochem., 26, 1287–1297 (1994).
947
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