Báo cáo Y học: Amphipathic property of free thiol group contributes to an increase in the catalytic efficiency of carboxypeptidase Y pot

Amphipathic property of free thiol group contributes to an increase
in the catalytic efficiency of carboxypeptidase Y
Joji Mima, Giman Jung, Takuo Onizuka, Hiroshi Ueno and Rikimaru Hayashi
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan
Cys341 of carboxypeptidase Y, which constitutes one side of
the solvent-accessible surface of the S1 binding pocket, was
replaced with Gly, Ser, Asp, Val, Phe or His by site-directed
mutagenesis. Kinetic analysis, using Cbz-dipeptide sub-
strates, revealed that polar amino acids at the 341 position
increased K
m
whereas hydrophobic amino acids in this
position tended to decrease K
m
. This suggests the involve-
ment of Cys341 in the formation of the Michaelis complex in
which Cys341 favors the formation of hydrophobic inter-
actions with the P1 side chain of the substrate as well as with
residues comprising the surface of the S1 binding pocket.
Furthermore, C341G and C341S mutants had significantly
higher k
cat
values with substrates containing the hydropho-
bic P1 side chain than C341V or C341F. This indicates that
the nonhydrophobic property conferred by Gly or Ser gives
flexibility or instability to the S1 pocket, which contributes to
the increased k
cat
values of C341G or C341S. The results
suggest that Cys341 may interact with His397 during cata-
lysis. Therefore, we propose a dual role for Cys341: (a) its

hydrophobicity allows it to participate in the formation of
the Michaelis complex with hydrophobic substrates, where it
maintains an unfavorable steric constraint in the S1 subsite;
(b) its interaction with the imidazole ring of His397 contri-
butes to the rate enhancement by stabilizing the tetrahedral
intermediate in the transition state.
Keywords: amphipathic property; carboxypeptidase Y;
substrate-binding site; tetrahedral intermediate; thiol group.
Carboxypeptidase Y from Saccharomyces cerevisiae,which
is localized in the vacuole and involved in the C-terminal
processing of peptides and proteins, belongs to the serine
carboxypeptidase family. It has a catalytic triad (Ser, His,
Asp) which constructs the charge-relay system at the active
center and exhibits peptidase and esterase activities with
broad substrate specificity [1–4]. Chemical modification
studies have assigned the essential serine and histidine
residues to positions 146 and 397, respectively [1–3]. A third
member of the catalytic center, aspartic acid, was putatively
assigned to position 338 based on structural homology with
carboxypeptidase II from wheat (CPW-II) [4].
Cys341 is the only one of the 11 Cys residues of
carboxypeptidase Y that is present as a free thiol group.
This single cysteine residue is conserved among the single-
chain serine carboxypeptidases; however, its role in the
catalytic mechanism and substrate binding remains unclear
and it has been the target of various chemical modification
studies [1,5,6]. Iodoacetate and iodoacetamide do not react
with Cys341 unless the enzyme is denatured [1]. On the
other hand, p-hydroxymercuribenzoate is able to react with
Cys341 to give an inactive enzyme that is no longer reactive

with di-isopropyl phosphorofluoridate, a modifier of the
essential Ser146. The effects of alkyl and aromatic mercurial
compounds on carboxypeptidase Y activity have raised the
question whether or not Cys341 plays an essential role in the
hydrolytic reaction [5,6].
Attempts have been made to clarify the role of Cys341 by
using site-directed mutagenesis techniques [7,8]. Winther &
Breddam [7] prepared mutant enzymes in which Cys341 was
replaced by Ser, Gly, Gln, Glu, His or Lys. A number of
their mutant enzymes exhibited reduced activity toward a
wide range of dipeptide and ester substrates.
These chemical modification and site-directed mutagen-
esis studies indicate that Cys341 is located at the substrate-
binding site in the hydrophobic environment, and also
suggest that Cys341 may be involved in the catalysis in a
manner other than substrate binding. However, the precise
type of catalytic event affected by Cys341 remains unclear.
X-ray crystallographic studies of Endrizzi et al. [9] have
shown that carboxypeptidase Y contains clearly defined
substrate-binding sites, S1¢ and S1 [10], each of which binds
the C-terminal (P1¢) and penultimate (P1) side chain of the
substrate, respectively. The S1¢ and S1 subsites are located
between two hydrophobic depressions separated by a
Ser146–His397 diad. The crystal structure of p-chloromer-
curibenzoic acid-modified carboxypeptidase Y has also
revealed that Cys341, along with other hydrophobic
residues, comprises the solvent-exposed surface in the S1
subsite. The other residues include Tyr147, Leu178, Tyr185,
Tyr188, Trp312, and Ile340 [9] (Fig. 1). The SH group of
Cys341 is also situated in the vicinity of the side chains of

Leu178, Ile340, and the essential His397 [9].
In these previous studies, the importance of the hydro-
phobic property of Cys341 has been neglected. In general,
when an SH group is fully protonated, it is able to form
hydrophobic interactions with neighboring hydrophobic
residues and aids in creating a hydrophobic environment
[11,12]. To address this issue, we constructed six mutants by
site-directed mutagenesis, in which Val and Phe mutants are
Correspondence to J. Mima, Division of Applied Life Sciences,
Graduate School of Agriculture, Kyoto University, Kitashirakawa,
Sakyo-ku, Kyoto 606-8502, Japan.
Fax: + 81 75 7536128, Tel.: + 81 75 7536125,
E-mail:
Enzyme: carboxypeptidase Y (EC 3.4.16.5).
(Received 15 January 2002, revised 13 May 2002,
accepted 15 May 2002)
Eur. J. Biochem. 269, 3220–3225 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02997.x
designed to maintain hydrophobicity and Gly, Ser, Asp and
His mutants to provide hydrophilicity. The catalytic roles of
Cys341 in carboxypeptidase Y are examined by comparing
the kinetic parameters of the Cys341 mutant enzymes.
MATERIALS AND METHODS
Materials
Cbz-Phe-Leu-OH was obtained from Fluka Chemie AG,
Buchs, Switzerland. Cbz-Ala-Phe-OH was from Sigma
Chemical Company, St Louis, MO, USA. Cbz-Gly-Phe-OH
and Cbz-Gly-Leu-OH were from the Peptide Institute Inc.,
Osaka, Japan. The synthetic oligonucleotides were obtained
from Japan Bio Services, Saitama, Japan. Restriction
endonucleases and T4 polynucleotide kinase were purchased

from Toyobo, Osaka, Japan. The Transformer
TM
site-
directed mutagenesis kit was purchased from Clontech, Palo
Alto, CA, USA. The Taq dyedeoxy
TM
terminator cycle
sequencing kit was obtained from Applied Biosystems,
Foster City, CA, USA. DEAE-Sephadex A-50 was from
Pharmacia Fine Chemicals, Uppsala, Sweden. Hydroxyl-
apatite gel was purchased from Bio-Rad, Hercules, CA,
USA. Bistris was obtained from Nacalai Tesque, Kyoto,
Japan. All other chemicals were of reagent grade and
obtained locally.
Strains and plasmid DNA
The plasmid pTSY3 containing the PRC1 gene coding
for carboxypeptidase Y and S. cerevisiae SEY2202
(MATa Dprc1::(LEU2) leu2-3,112ura3-52hi4-519)were
kindly provided by Dr Klaus Breddam, Carlsberg Labor-
atory, Copenhagen, Denmark. Escherichia coli JM109
(recA1 supE44 endA1 hsdR17 gyrA96 relA1 thiD(lac-proAB)
F¢ [traD36 proAB
+
laqI9 lacZDM15]) was from the
in-house collection.
Site-directed mutagenesis
In vitro mutagenesis was performed with the pTSY3
subclone of the PRC1 gene [13]. Three oligonucleotides
were used for mutation: a 25-mer as the mutagenic primer to
introduce other amino acids (Gly/His/Ser/Val/Asp/Phe) for

Cys341 (sequence 5¢-AAGATTTCATCGGT/CAT/TCT/
GTT/GAT/TTCAACTGGTTGGG-3¢);a26-merasthe
selection primer (5¢-ACTACAAAATGAGCTCCCTCGC
GCGT-3¢) to introduce a PvuII restriction site into plasmid
pTSY3 for selection; and a 20-mer sequence primer for
DNA sequencing (5¢-GCCATGGAAGTACGACGA
AG-3¢). The mutation was performed with a Transformer
site-directed mutagenesis kit as described by Deng &
Nickoloff [14]. The DNA sequence reaction was performed
with a Taq dyedeoxy terminator cycle sequencing kit. Yeast
strain SEY2202 was transformed by the lithium acetate
method [15]. E.coliJM109 was transformed by the method
of Hanahan [16] using a standard transformation buffer.
Purification of wild-type and mutant forms
of carboxypeptidase Y
Typical purifications were carried out as previously des-
cribed [17]. An additional purification step of hydroxyl-
apatite column chromatography was performed essentially
as described by Bernardi [18]. The preparations, at 4 °C,
were loaded on to a hydroxylapatite column equilibrated
with 75 m
M
sodium phosphate buffer, pH 6.8, and both
wild-type carboxypeptidase Y and mutant carboxypepti-
dase Y were eluted with 150 m
M
buffer. The enzyme
solutions were desalted on ultrafiltration apparatus
(Amicon stirred cells model 8050) by repeated concentration
and dilution with water. Enzyme activity was assayed with

N-benzoyl-
L
-tyrosine-p-nitroanilide during the purification
steps [19]. The purity of the refined enzymes was verified by
SDS/PAGE.
CD measurements
CD spectra were measured on a JASCO J-720 W spectro-
polarimeter at room temperature. Ten scans were averaged
for the wild-type and mutant forms of carboxypeptidase Y
at concentrations of % 3.0 l
M
in 10 m
M
sodium phosphate,
pH 7.0.
Kinetic characterization
Peptidase activities were measured in 50 m
M
Bistris
buffer/1 m
M
EDTA,pH6.5,at25°C. Enzyme concen-
trations of the wild-type and mutant carboxypeptidase Y
in the hydrolysis reaction were 32.8 n
M
. Substrate con-
centrations were 0.02–20 m
M
. The reaction was termin-
ated and deproteinized by adding 330 lL0.4

M
sulfosalicylic acid per mL of the enzyme/substrate solu-
tion. The initial rates of hydrolysis of dipeptide substrates
were measured by quantitating the amount of C-terminal
amino acid released, using a Jeol JLC-300 amino-acid
analyzer. The kinetic parameters for the hydrolysis of
various Cbz-peptide substrates were derived from Hanes-
Woolf plots.
Fig. 1. Catalytic triad (Ser146, His397, and Asp338) and S1 binding site
of carboxypeptidase Y. Cys341 constitutes the left side of the solvent-
accessible surface with Ile340 viewed from the point of the active Ser-
Hisdiad.ThesulfuratomofCys341islocatedwithin5A
˚
of the
imidazole ring of His397.
Ó FEBS 2002 A single thiol group in carboxypeptidase Y (Eur. J. Biochem. 269) 3221
Calculation of energy levels of intermediates
and apparent binding energies
The hydrolysis of the peptide substrates catalyzed by
carboxypeptidase Y is described in Scheme 1. For typical
serine protease catalysis, an acylation step is the rate-
determining step (k
2
( k
3
); therefore, K
m
% K
s
and

k
2
% k
cat
are assumed [20]. The energy level, DG,ofeach
enzyme state was calculated from the following thermody-
namic equations [20] and is shown relative to the free
enzyme defined as DG ¼ 0(Table3):
DG
s
¼ RT lnK
s
% RT lnK
m
;
DG
‡
¼ RT lnðk
B
T=hÞÀRT lnk
2
% RT lnðk
B
T=hÞÀRT lnk
cat
;
DG
‡
T
¼ RT lnðk

B
T=hÞÀRT lnðk
2
=K
s
Þ
% RT lnðk
B
T=hÞÀRT lnðk
cat
=K
m
Þ;
DDG
‡
¼ DG
‡
ðmutantÞÀDG
‡
ðwild-typeÞ
where DG
s
is the binding energy of the substrate to the
enzyme, DG
à
is the activation energy in the chemical steps of
bond making and breaking, DG
‡
T
is the activation energy for

the free enzyme reacting with the free substrate to give
products, R is the gas constant, T is the absolute tempera-
ture, k
B
is the Boltzmann’s constant, h is Planck’s constant.
RESULTS
Purification of mutant carboxypeptidase Y
Mutant carboxypeptidase Y in which Cys341 is replaced by
Gly, Ser, Asp, Val, Phe, or His residues was isolated and
purified from % 150 g yeast cells (Table 1). Single bands on
SDS/PAGE analysis verified the purity of all mutants. The
protein yields of C341G, C341S and C341D were similar to
that of the wild-type, whereas the yields of C341V, C341F
and C341H were lower. The specific activities of all the
mutants were decreased by more than 10-fold compared
with the wild-type, with C341D and C341F showing more
than a 100-fold decrease and C341H was nearly inactive.
Properties of mutant carboxypeptidase Y
The effect of the amino-acid substitution at Cys341 on
secondary structure was evaluated by analyzing the CD
spectra (Fig. 2). All mutant enzymes except C341H had a
spectrum identical with that of the wild-type enzyme. The
different CD spectrum for C341H suggests that the
introduction of a positive charge at 341 causes some
alteration in the secondary structure. Because of the poor
yield and altered CD spectrum, C341H mutant was not
analyzed further.
Kinetic properties of mutant carboxypeptidase Y
The effects of amino-acid substitution on the enzymatic
properties of the Cys341 mutant enzymes were investigated

using two sets of substrates, Cbz-X
1
-Leu-OH and Cbz-X
2
-
Phe-OH, where X
1
was a Gly or Phe residue and X
2
was a
Gly or Ala residue (Table 2). Mutant enzymes had reduced
k
cat
/K
m
values with all four substrates compared with the
wild-type enzyme, except that C341G had a somewhat
increased value with Cbz-Ala-Phe.
Mutant enzymes exhibited similar k
cat
/K
m
values with
Cbz-Gly-Leu and Cbz-Gly-Phe, although C341G exhibited
higher k
cat
/K
m
values than the other mutants with Cbz-Gly-
Leu. The k

cat
values of C341G, C341S and C341F were
largely higher than that of the wild-type. A nearly identical
profile was observed for K
m
values with Cbz-Gly-Leu and
Scheme 1.
Table 1. Yields of wild-type carboxypeptidase Y and Cys341 mutants
on purification from 150 g yeast cells. Enzymes were purified by the
method of Hayashi et al. [17]. Activity toward N-benzoyl-
L
-tyrosine-
p-nitroanilide was determined [19]. ND, not detected.
Enzyme
Total protein
(mg)
Total activity
(units)
10
)3
· Specific
activity (unitsÆmg
)1
)
Wild-type 3.9 3.4 860
C341G 3.3 0.24 72
C341S 4.9 0.31 62
C341D 4.6 0.014 3.0
C341V 1.0 0.032 32
C341F 1.0 0.0053 5.1

C341H 0.41 ND –
Fig. 2. CD spectra of wild-type carboxypeptidase Y and Cys341
mutants. Protein concentrations of carboxypeptidase Y and its
mutants were % 3.0 l
M
in 10 m
M
sodium phoshate, pH 7.0. Condi-
tions for measurements: band width 1.0 nm; sensitivity 50 millidegrees;
response 0.5 s; wave length 190–250 nm; scan speed 100 nmÆmin
)1
;
step resolution 1 nm; 10 measurements were made. (d) Wild-type
carboxypeptidase Y; (m) C341G; (h) C341S; (j) C341D; (r) C341V;
(s) C341F; (e) C341H.
3222 J. Mima et al.(Eur. J. Biochem. 269) Ó FEBS 2002
Cbz-Gly-Phe. This suggests that, when glycine occupies the
S1 subsite, the binding preference of the S1¢ subsite for
hydrophobic amino acid is not absolute, but is still affected
by the mutation at position 341.
Mutant enzymes with a hydrophobic residue at the 341
position, i.e. C341V and C341F, had lower K
m
values with
Cbz-Phe-Leu and Cbz-Ala-Leu than the wild-type enzyme.
This suggests that a hydrophobic interaction at the S1
pocket is important for substrate binding. On the other
hand, hydrophobic residues at position 341 significantly
decreased the k
cat

values (20–90-fold decreased compared
with the k
cat
value of the wild-type carboxypeptidase Y),
while values for C341G and C341S were decreased only
1.5-fold to sixfold over that of the wild-type enzyme.
C341G exhibited higher k
cat
/K
m
values with all substrates
than the other mutants. This value was even higher than the
wild-type when Cbz-Ala-Phe was used as a substrate. The
characteristics of the C341S mutant were similar to those of
C341G but its binding ability was slightly less. Both C341G
and C341S maintained a relatively high enzyme activity.
These results for C341G and C341S with hydrophobic
dipeptide substrates are in agreement with the results
obtained in previous work [7].
DISCUSSION
Effect of replacement of Cys341 on kinetic constants
The introduction of Gly at position 341 would be expected
to reduce any structural constraints at the S1 subsite because
of elimination of the bulky SH group. It has been suggested
that eliminating steric constraints present in the S1 subsite of
the wild-type carboxypeptidase Y would increase the activ-
ity of the Leu178 mutant carboxypeptidase Y toward
substrates with the basic P1 side chains [21]. It was assumed
that the lack of a side chain at position 341 would make the
S1 pocket unstable because of the elasticity introduced. The

size of the S1 subsite may also be reduced as the result of
shrinkage of the hydrophobic side chains in the surface of
the S1 pocket which is exposed to solvent. We were able to
engineer the P1 preference of carboxypeptidase Y from a
bulky hydrophobic side chain, i.e. Phe, to a small hydro-
phobic residue, i.e. Ala, on the C341G mutant carboxy-
peptidase Y. It was evident that the k
cat
/K
m
value with
Cbz-Ala-Phe-OH increases up to 556-fold relative to the
hydrolysis of Cbz-Gly-Phe-OH, whereas only a 27-fold
increase was obtained with Cbz-Phe-Leu-OH relative to
Cbz-Gly-Leu-OH (Table 2). It is also shown that C341G
narrows the P1 amino-acid preference as it no longer
possesses a wild-type-like preference for a bulky hydropho-
bic amino acid. Of the mutant enzymes, C341G had the
highest k
cat
values with respect to substrates Cbz-Gly-Leu
and Cbz-Ala-Phe (Table 2). Although detailed structural
evaluation is needed, the reason for C341G exhibiting high
k
cat
values is probably the increased elasticity at the S1
subsite.
C341S behaves in a similar manner to C341G in its
kinetic profile, except that it tends to have higher K
m

.This
low affinity of C341S can be attributed to the water
molecule(s) co-ordinated to the solvent-accessible surface of
the S1 subsite via the hydroxy group. This additional
interaction with water molecule(s) may inhibit the forma-
tion of the Michaelis complex (Scheme 1), which results in
increased K
m
values (Table 2). It was also assumed in a
previous report [7] that the higher K
m
values of C341S are
due to co-ordination of water molecule(s) around the
hydrophilic side chain. When the Michaelis complex is
formed with substrates that have hydrophobic P1 side
chains, the surface of the S1 subsite may become inaccessible
to the solvent, which would cause reorientation of the side
chain of Ser directly away from the hydrophobic surface of
the S1 subsite. In the transition state, the steric environment
of the S1 pocket in C341S may be almost identical with that
of C341G, which explains the similar catalytic characteris-
tics of C341S and C341G (Table 2).
A hydrophilic group, such as Asp or Ser, at the S1 subsite
has a tendency to reduce its affinity for substrates. The
substitution of the Cys341 with a negatively charged amino
acid led to an increase in K
m
. Mutant enzymes in which
Cys341 was replaced with Glu and Gln had higher K
m

values than wild-type carboxypeptidase Y [7]. This suggests
that formation of hydrogen bonds or electrostatic interac-
tions at the S1 subsite may not be involved in the substrate-
recognition mechanism.
In general, a thiol group does not undergo deprotona-
tion when it is in a hydrophobic environment [11,12].
Therefore, we postulate that Cys341, with a fully proto-
nated SH group, maintains hydrophobic interactions with
neighboring amino-acid residues. C341V, which has a
hydrophobic residue almost identical with cysteine in size
at position 341, would be predicted to show kinetic
constants similar to the wild-type carboxypeptidase Y.
Indeed our results show that C341V and wild-type
Table 2. Kinetic parameters of wild-type carboxypeptidase Y and
Cys341 mutants for hydrolysis of Cbz-dipeptide. ND, not determined
because K
m
values exceeded the applicable substrate concentration
range.
Enzyme Substrate
k
cat
(s
)1
)
K
m
(m
M
)

k
cat
/K
m
(s
)1
Æm
M
)1
)
Wild-type Cbz-Gly-Leu 3.2 0.83 3.8
Cbz-Phe-Leu 92 0.19 480
Cbz-Gly-Phe 0.96 0.41 2.3
Cbz-Ala-Phe 150 1.1 140
C341G Cbz-Gly-Leu 7.0 3.2 2.2
Cbz-Phe-Leu 15 0.26 59
Cbz-Gly-Phe 1.1 3.7 0.30
Cbz-Ala-Phe 95 0.57 170
C341S Cbz-Gly-Leu 1.9 5.2 0.37
Cbz-Phe-Leu 18 0.32 57
Cbz-Gly-Phe 2.4 6.7 0.36
Cbz-Ala-Phe 93 4.2 22
C341D Cbz-Gly-Leu 1.4 9.1 0.15
Cbz-Phe-Leu 2.1 3.0 0.71
Cbz-Gly-Phe ND ND 0.15
Cbz-Ala-Phe ND ND 3.4
C341V Cbz-Gly-Leu 0.48 0.89 0.54
Cbz-Phe-Leu 4.6 0.16 29
Cbz-Gly-Phe 0.16 0.45 0.36
Cbz-Ala-Phe 8.0 1.1 7.5

C341F Cbz-Gly-Leu 2.3 8.4 0.27
Cbz-Phe-Leu 1.2 0.070 25
Cbz-Gly-Phe 5.5 19 0.28
Cbz-Ala-Phe 2.2 0.98 2.3
Ó FEBS 2002 A single thiol group in carboxypeptidase Y (Eur. J. Biochem. 269) 3223
carboxypeptidase Y have similar K
m
values with all
substrates examined (Table 2). This provides support for
the hypothesis that Cys341 is not only located at the S1
binding site [5–7,9], but the hydrophobicity of the thiol
group also plays a role in substrate binding and
interacting with the hydrophobic residues of the S1
subsite. However, the k
cat
values of C341V were much
lower than those of the other mutant carboxypeptidase Y,
i.e. C341G or C341S (Table 2). It is possible that the
hydrophobic interaction of Val with other side chains in
the S1 pocket or P1 side chain of the substrates in the
Michaelis complex inhibits the acylation step in the
hydrolysis reaction (Scheme 1). It can be assumed that
the hydrophobic interaction of Val in the S1 subsite is
stabilized so that the rate of acylation becomes signifi-
cantly reduced. In the case of the mutant enzymes with
bulky hydrophobic residues at position 341, i.e. C341F,
the hydrophobicity at position 341 appears to be import-
ant for substrate binding, although it may not necessarily
have any effect on the rate of acylation.
Roles of Cys341 in the catalytic mechanism

of carboxypeptidase Y
Before the formation of the Michaelis complex, the free
thiol group of Cys341 participates in a hydrophobic bond
network on the solvent-accessible surface of the S1 subsite,
which is constructed of Tyr147, Leu178, Tyr185, Tyr188,
Trp312, and Ile340 (Fig. 1). Thus, the thiol group of Cys341
may participate in controlling the depth and width of the S1
solvent-accessible cavity, where a bulky hydrophobic P1
side chain such as phenylalanine can be accommodated. At
the time the Michaelis complex is established, the free thiol
group of Cys341 is located in close proximity to the P1 side
chain and the hydrophobic interaction with hydrophobic P1
side chain is in effect. In fact, our results for C341V and
C341F provide support for a scenario in which the
hydrophobicity of the SH group of Cys341 is important
for substrate binding and maintaining the solvent-accessible
cavity at the S1 subsite.
However, in the case of the transition state in the
acylation reaction, the role of Cys341 as a hydrophobic
residue should be altered because C341V, which is solely
hydrophobic in nature, exhibits a lowered k
cat
. This suggests
an additional role for this thiol group.
Free-energy parameters derived from the kinetic analysis
aresummarizedinTable3forthewild-typeandC341V
mutant enzymes. This result can be visualized in the form of
a diagrammatic scheme shown in Fig. 3. A characteristic of
C341V is its increased activation energy, DG
à

, at the stage
where the tetrahedral intermediate is formed, the step that
has the most influence on the rate of acyl enzyme formation.
We are concerned as to why C341V exhibits an increased
DG
à
compared with the wild-type enzyme. As the properties
of Val, such as size and hydrophobicity, are similar to those
of Cys and the affinity of C341V for the substrates tested are
similar to that of the wild-type enzyme, there must be some
explanation for the difference in DG
à
.
What is the specific role of Cys341 in the transition state?
In the tetrahedral transition state, Cys341 is located adjacent
to His397 of the catalytic center [9], the imidazole nitrogen
of which is positively charged (Fig. 4). The thiol group of
Cys341 is located within 5 A
˚
of the imidazole nitrogen of
His397. Therefore, it is reasonable to assume that the sulfur
atom of Cys341 becomes polarized by the positive charge on
the imidazole nitrogen of His397. This newly created
electrostatic interaction may cause redirection of the cystei-
nyl side chain from the surface of the S1 subsite toward the
charged imidazole nitrogen (Fig. 4).
SuchanalterationinthesidechainofCys341inthe
tetrahedral intermediate may give structural flexibility to the
S1 subsite, which reduces steric constraint at the S1 subsite:
elimination of the side chain of Cys341 from the S1 solvent-

accessible surface weakens a hydrophobic bond network,
which maintains the depth and width of the S1 subsite cavity.
The flexibility introduced wouldbe used to create asubstrate-
Table 3. Gibbs free energies (kJÆmol
)1
) of complexes of wild-type
carboxypeptidase Y and C341V mutant. DG
S
is algebraically negative
and DG
à
and DG
‡
T
positive. The activation energy (DG
à
) of wild-type
carboxypeptidase Y is lowered by DDG
à
compared with that of
C341V.
Substrate Enzyme DG
‡
s
DG
à
DG
‡
T
DDG

à
Cbz-Phe-Leu Wild-type )21 62 40 –
C341V )22 69 48 7.5
Cbz-Ala-Phe Wild-type )17 59 41 –
C341V )17 66 49 7.5
Fig. 3. Free-energy profiles for the formation of the tetrahedral inter-
mediate by wild-type (energy level in heavy line) and C341V (in light line)
carboxypeptidase Y.
Fig. 4. Model of alteration in the S1 subsite in the transition state
through interaction between Cys341 and His397 making it comple-
mentary with the P1 side chain of the substrate.
3224 J. Mima et al.(Eur. J. Biochem. 269) Ó FEBS 2002
binding pocket that is complementary in size to various
hydrophobic P1 amino acids (Fig. 4) and thereby increase
the interaction energy of the tetrahedral intermediate with
the substrate. Because C341V lacks the ability to interact
with the protonated His397, this mutant enzyme is inefficient
at utilizing the increased interaction energy to stabilize the
tetrahedral intermediate in the transition state (Fig. 3).
The described flip-flap motion of Cys341 explains our
experimental data and leads us to propose that Cys341 may
have two distinct roles in the catalytic mechanism: (a) the
hydrophobicity of the thiol group (Cys341) is involved in
substrate binding at the S1 subsite and in maintaining the
width and depth of the S1 subsite; (b) the rearrangement of
the S1 subsite induced by the interaction between the SH
group and the imidazole nitrogen of His397 stabilizes the
transition state. X-ray crystallography of the mutant
enzymes and their complexes with the transition state
analog to confirm the hypothesis of a dual role for Cys341 is

underway.
We suggest that the proposed function is common to free
thiol groups adjacent to active histidine residues found in
the monomeric serine carboxypeptidases including carb-
oxypeptidase S1 from Penicillium janthinellum, carboxy-
peptidase MIII from barley malt, and Kex1p from
S. cerevisiae [22]. As a number of subtilisin-like serine
endoproteases, e.g. proteinase B from S. cerevisiae,prot-
einase K from Tritirachium album, and thermitase from
Thermoactinomyces vulgaris, have free thiol groups in the
vicinity of the catalytic triad (Ser, His, Asp) [7,23], these
cysteine residues may also have the putative dual role in the
hydrolysis reactions. The present hypothesis provides fur-
ther insights, with the revelation of the new residues at
subsites that contribute to catalytic efficiency in the
transition state.
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Ó FEBS 2002 A single thiol group in carboxypeptidase Y (Eur. J. Biochem. 269) 3225