Relationship between the hydrophobicity of dipeptides and the Michaelis-Menten constant Km of their hydrolysis by carboxypeptidase-Y and carboxypeptidase-A

Article abstract of DOI:10.1246/bcsj.77.1187

The enzymatic hydrolysis of dipeptides by carboxypeptidase-Y and carboxypeptidase-A was investigated. In the enzymatic hydrolysis of the dipeptides, a good linear relationship (r = 0.997 and 0.999) was found between the Michaelis-Menten constant (K_m) and the hydrophobicity of the substrates evaluated from relative elution volume in reversed-phase HPLC. The correlation suggests that the hydrophobicity of the C-terminal amino acid is a major factor in governing the stability of the enzyme-substrate complex. The difference in the slope of the linear-regression lines seems to reflect the degree of relative hydrophobicity of the binding pockets in carboxypeptidase-Y and carboxypeptidase-A.

Full text of DOI:10.1246/bcsj.77.1187

ꢀ 2004 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 77, 1187–1193 (2004) 1187
Relationship between the Hydrophobicity of Dipeptides and
the Michaelis–Menten Constant Km of Their Hydrolysis
by Carboxypeptidase-Y and Carboxypeptidase-A
ꢀ
Yoshifumi Kanosue, Satoshi Kojima,1 Yoshikazu Hiraga, and Katsuo Ohkata
Department of Chemistry, Graduate School of Science, Hiroshima University,
1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526
1Department of Chemistry, Graduate School of Science, and Center for Quantum Life Sciences,
Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526
Received November 11, 2003; E-mail: ohkata@sci.hiroshima-u.ac.jp
The enzymatic hydrolysis of dipeptides by carboxypeptidase-Y and carboxypeptidase-A was investigated. In the en-
zymatic hydrolysis of the dipeptides, a good linear relationship (r ¼ 0:997 and 0.999) was found between the Michaelis–
Menten constant (Km) and the hydrophobicity of the substrates evaluated from relative elution volume in reversed-phase
HPLC. The correlation suggests that the hydrophobicity of the C-terminal amino acid is a major factor in governing the
stability of the enzyme–substrate complex. The difference in the slope of the linear-regression lines seems to reflect the
degree of relative hydrophobicity of the binding pockets in carboxypeptidase-Y and carboxypeptidase-A.
The hydrophobicity of solutes often influences various bio-
logical phenomena as well as chemical properties.1,2 Hydro-
phobicity is a characteristic of materials that indicates its disfa-
vor of water. Solutes high in this character tend to be expelled
from water structure. Arising from our interest in the relation-
ship between the hydrophobicity of organic compounds and
biological or chemical properties,3,4 we have carried out an ex-
amination of the hydrophobicity of several dipeptides and their
enzymatic hydrolysis by carboxypeptidase-Y and carboxypep-
tidase-A. In enzymatic reactions, the hydrophobicity of sub-
strates often avails to incorporate them into enzymes from
aqueous media.
Carboxypeptidase-Y (CPD-Y) [EC 3.4.16.5] from bakers
yeast is a lysosomal serine exopeptidase, which catalyzes the
hydrolysis of the carboxyl-terminal peptide bond of proteins.
It has been suggested that a Ser–His–Asp catalytic triad is in-
volved in the process, as found in the trypsin and chymotrypsin
families of endopeptidases.5 An interesting feature is that a pep-
tide bond consisting of a proline residue can be hydrolyzed by
the catalysis of CPD-Y, while this bond is resistant to cleavage
by ꢀ-chymotrypsin and carboxypeptidase-A (CPD-A) [EC
3.4.17.1]. CPD-Y is also a popular model protein for studying
protein sorting in yeast.6 CPD-A from bovine pancreas is a zinc
exopeptidase, and is also known to catalyze the hydrolysis of
the carboxyl-terminal peptide bond of proteins. The catalysis
reaction occurs when the peptide carbonyl group is coordinated
to zinc, and a water molecule is delivered to the substrate with
catalytic Glu assisting as a general base.7
In general, enzyme catalysis is initiated by the interaction be-
tween the enzyme and the substrate prior to reaction, which
gives rise to what is known as the enzyme–substrate (E–S)
complex or Michaelis complex as shown in Scheme 1. The
stability of the E–S complex is related to the affinity of the sub-
strate to the enzyme, which is measured by the Michaelis–
Menten constant (Km) for the E–S complex. Generally, the
Km values are affected by various types of weak non-covalent
interactions involved in the formation of the E–S complex
(electrostatic interactions, hydrogen bonds, and hydrophobic
interactions, etc.). Noteworthy among them are usually weak
hydrophobic interactions, which have been suggested to be
important not only for the formation of the E–S complex
(Michaelis complex), but also substrate discrimination.8 Since
the complex between the substrate and the enzyme is formed
with weak noncovalent interactions, the reaction between
enzyme and substrate is reversible; this phenomenon is very
important for product release.
As a step towards understanding the influence of the hydro-
phobicity of substrates in the hydrolysis of dipeptides by en-
zymes, we have pursued this issue by comparing the difference
in the hydrolytic behavior of peptides catalyzed by CPD-Y and
Scheme 1.
Published on the web June 9, 2004; DOI 10.1246/bcsj.77.1187

1188 Bull. Chem. Soc. Jpn., 77, No. 6 (2004)
Hydrolysis of Dipeptides by CPD-Y and CPD-A
where ’ is the phase ratio of the column. The energetics of re-
tention is determined by the standard free-energy change
(ꢀGret) associated with solute transfer from the mobile phase
to the stationary phase, by
ꢀGret ¼ ꢁRT ln K:
ð2Þ
The relative free energy (ꢁꢀGret) of partition is derived from
ꢁꢀGret ¼ ꢁRT lnðKR=KSÞ
¼ ꢁRT½lnðVe ꢁ V0ÞR ꢁ lnðVe ꢁ V0ÞSꢂ;
ð3Þ
where KR=KS is the equilibrium constant relative to that (KS) of
the standard compound (in this case, Z–Phe–Gly), Ve is the elu-
tion volume, and V0 is the column volume in HPLC. The values
of Ve ꢁ V0 can be evaluated from the corresponding retention
times, tr and t0. Combining Eqs. 1 and 3 yields
ꢁꢀGret ¼ ꢁRT lnðk0R=k0SÞ ¼ ꢁ2:303RTðꢁlog k0Þ; ð4Þ
where k0R=k0S is the capacity factor relative to that of the stand-
ard compound (in this case, Z–Phe–Gly). Thus, ꢁlog k0 is the
difference in the log k0 values with that of the standard com-
pound (in this case, Z–Phe–Gly).
It has been demonstrated by Pliska and his co-workers that
the hydrophobic parameters, log P for ꢀ-amino acids, are high-
ly correlated to the Rf values of thin-layer chromatography on
silica gel and cellulose plates,11 which has been revealed like-
wise by others for ODS columns.12
Regarding conditions, the column used was a 250 ꢃ 4:6 mm
ODS reversed-phase column (Wakosil II 5C18HG), and the
mobile phase (0.5 mL/min) consisted of acetonitrile–water
(3:7) containing 60 mM AcONa buffer (pH 6.0). In order to
confirm the validity of using ꢁꢀGret, we first examined the re-
lationship between ꢁꢀGret and log P for Gly, Ala, Pro, Tyr, and
Phe, and observed good linearity, as follows:
Scheme 2.
CPD-A in relation with substrate hydrophobicity, as described
herein.
ꢁꢀGret ¼ ꢁ0:503 log P ꢁ 3:01 ðr ¼ 0:954; n ¼ 5Þ: ð5Þ
ꢁꢀGret values were also measured with a slightly different sol-
vent composition of acetonitrile–water = 23:77. Although the
absolute retention times were much longer for this mobile
phase (50–70 min compared with 15–25 min), a good relation-
ship (correlation factor r ¼ 0:983) was found between the two
sets of ꢁꢀGret values for the two mobile phases of differing
composition. Thus, although the absolute retention time could
change with differing mobile-phase composition, the relative
free-energy relationship between the ꢁꢀGret and Km values to
be evaluated (vide infra) are expected to be the same. The rel-
ative free-energy values of partition (ꢁꢀGret) for the dipeptides
are summarized in Table 1.
Kinetics. All of the kinetic experiments were performed in
pH 6.5 solutions at 37 ꢄC. The progress of the reactions was de-
termined by spectrophotomerically (230–240 nm) monitoring
the relative amount of the released products. The kinetic param-
eters (Km, Ki, and k2), as designated in Scheme 1 for the hydrol-
ysis of these dipeptides or for the inhibition of Z–Phe–L-Pro hy-
drolysis by peptides bearing a D-amino acid by CPD-Y and
CPD-A, are summarized in Table 1. The listed values are the
average of 3–5 runs. The kinetic data was calculated by non-lin-
ear fitting of the Michaelis–Menten equation to plotted experi-
mental data.
Results and Discussion
Preparation and Hydrolysis of N-Benzyloxycarbonyl
Protected Dipeptides. A series of dipeptides [Z–Phe–Gly,
Z–Phe–Sar, Z–Phe–D-Ala, Z–Phe–L-Ala, Z–Phe–NMeAla, Z–
Phe–Aib, Z–Phe–D-Pro, Z–Phe–L-Pro, and Z–Phe–Phe(p-X,
X = H, OH, OMe)], as shown in Scheme 2, were prepared
by way of typical procedures.
Evaluation of Hydrophobicity by Means of a Chromato-
graphic Method. The hydrophobicities of the dipeptides were
evaluated as their affinity towards the stationary phase in re-
versed-phase HPLC relative to elution volume. In reversed-
phase chromatography with ODS columns, the retention of a
solute (S), i.e., a dipeptide, is viewed as the reversible associa-
tion of the solute with an octadecyl function (L) at the chroma-
tographic surface, thus forming a complex (SL). Solute reten-
tion is governed by the average value of the contact surface area
arising from the plurality of such binding configurations.9,10
The capacity factor (k0) is related to the equilibrium constant
(K) for the distribution of the solute between the bulk mobile
phase and the stationary phase by
k0 ¼ K’;
ð1Þ

Y. Kanosue et al.
Bull. Chem. Soc. Jpn., 77, No. 6 (2004) 1189
Table 1. Kinetic Parameters for Hydrolysis of Dipeptides by CPD-Y or CPD-A
aÞ
Dipeptide
ꢁꢀGret
CPD-Y
CPD-A
Z–Phe–Xaa
Km/mM
k2/sꢁ1
Km/mM
k2/sꢁ1
–NMeAla
–L-Pro
–L-Ala
–Sar
–Gly
–Aib
–L-Phe
–L-Phe(p-OH)
–L-Phe(p-OMe)
ꢁ0:677
ꢁ0:604
ꢁ0:293
ꢁ0:152
0.00
0:274 ꢅ 0:033
0:339 ꢅ 0:018
0:939 ꢅ 0:099
1:94 ꢅ 0:06
0:657 ꢅ 0:027
45:0 ꢅ 0:8
—
—
—
—
187:7 ꢅ 11:3
25:9 ꢅ 0:6
0:238 ꢅ 0:030
—
—
13:9 ꢅ 0:63
—
—
3:36 ꢅ 0:12
189:4 ꢅ 5:5
147:3 ꢅ 6:0
194:4 ꢅ 3:0
313:9 ꢅ 9:1
143:1 ꢅ 1:6
ꢁ1:28
ꢁ2:91
ꢁ0:854
ꢁ3:22
2:86 ꢅ 0:15
—
—
0:180 ꢅ 0:009
0:360 ꢅ 0:024
0:435 ꢅ 0:010
0:143 ꢅ 0:007
0:196 ꢅ 0:005
0:132 ꢅ 0:005
608:3 ꢅ 7:7
626:1 ꢅ 2:9
673:2 ꢅ 6:3
aÞ
ꢁꢀGret
Ki/mM
Inhibition type
–D-Pro
–D-Ala
ꢁ0:286
0:680 ꢅ 0:013
1:02 ꢅ 0:05
Competitive
Competitive
ꢁ0:444
a) ꢁꢀGret was calculated from the retention time in HPLC (ODS reversed-phase column).
Fig. 1. A linear relationship between log Km or log Ki and hydrophobic property (ꢁꢀGret) of the dipeptides. CPD-Y ( ), CPD-A
). Plots in the rectangles correspond to those of Ki.
(
Relationship between Km and Hydrophobic Properties of
Dipeptides. The order of the Km values was Phe < NMeAla <
L-Pro ; Phe(p-OH) < Phe(p-OMe) < L-Ala < Sar < Aib <
Gly in the hydrolysis by CPD-Y and Phe(p-OMe) < Phe <
Phe(p-OH) < L-Ala in the hydrolysis by CPD-A. Thus, the af-
finity of the Phe, NMeAla, and Pro substrates towards the CPD-
Y enzyme is more favorable for making the E–S complex rel-
ative to that of Ala, Sar, Aib, and Gly substrates and the affinity
of Phe(p-OMe), Phe, and Phe(p-OH) substrates towards CPD-
A is more favorable than that of Ala. On the other hand, Z–Phe–
D-Ala and Z–Phe–D-Pro caused the competitive inhibition of
Z–Phe–L-Pro hydrolysis with the Ki values being in the order
of Z–Phe–D-Ala > Z–Phe–D-Pro.
The k2 values of reactions involving CPD-Y [Phe(p-OH) >
Phe ; L-Ala > Gly > Aib ; Phe(p-OMe) > L-Pro > Sar >
NMeAla], show that N-unsubstituted amino acids were easily
hydrolyzed, whereas those of CPD-A reactions [Phe(p-OMe)
> Phe(p-OH) > Phe > L-Ala] indicated that aromatic amino
acids were easily hydrolyzed.
Thus, we decided to elucidate the relationship between hy-
drophobicity and Km or Ki for this case. There was found a lin-
ear free-energy relationship between log Km and ꢁꢀGret, which
reflects hydrophobic properties, as shown in Fig. 1. That is,
log Km ¼ 1:62ꢁꢀGret þ 0:51 (r ¼ 0:997, n ¼ 5) for CPD-Y
for dipeptide substrates [excluding those bearing amino acid
moieties of the D-series, and those bearing Aib, Phe, Phe(p-
OH), and Ph(p-OMe)], and log Km ¼ 0:0845ꢁꢀGret ꢁ 0:604;
r ¼ 0:999, n ¼ 4 for CPD-A.
The hydrolysis of N-alkyl substituted substrates and Z–Phe–
Aib by CPD-A was not observed, and the reaction of Z–Phe–
Gly was too slow to measure.
Because peptides of the D-series were much less reactive as

1190 Bull. Chem. Soc. Jpn., 77, No. 6 (2004)
Hydrolysis of Dipeptides by CPD-Y and CPD-A
substrates, their behavior as competitive inhibitors toward the
hydrolysis of the L-series was evaluated by non-linear fitting
of the Michaelis–Menten equation. Although the plots corre-
sponding to the two D-series dipeptides were slightly deviated
from the above linear line, as a whole, the Ki values of the D-
substrates were found to be colinear with the other five Km val-
ues according to the relationship, log Km ¼ 1:52ꢁꢀGret þ 0:48;
r ¼ 0:946, n ¼ 7 (including D-Ala and D-Pro). Thus, the corre-
lation between the hydrophobicity of the dipeptide substrates
and Km (along with Ki) elucidated in this study suggests that
the hydrophobicity of the C-terminal amino acid is a major fac-
tor in governing the stability of the enzyme–substrate and en-
zyme–inhibitor complex. The large upper deviation of the plot
of Z–Phe–Aib from the linear relationship implies that sterical
factors resulting from the geminal ꢀ-methyl groups inhibit in-
corporation of the substrate. Namely, as illustrated in Fig. 2, the
pro-R methyl group of Z–Phe–Aib apparently cannot fit into the
hydrophobic region of the S10 binding pocket of CPD-Y. For
the same reason, neither can the ꢀ-side chains of D-amino acids
in conformations required for the ensuing hydrolysis reactions.
Thus, positive hydrophobic interactions within the active site
expected from chromatographic considerations is diminished.
Judging from the extremely large upper side drift of the
log Km values from linearity for the Phe(p-X, X = H, OH,
OMe) substrates, although they are of the L-series, the size of
the pocket itself must be small. On the other hand, since the
space within the S10 binding pocket of CPD-A is presumably
sufficiently large enough to incorporate these aromatic groups,
the Km values of the substrates bearing these substituents show
a good correlation with that of L-Ala in relation with substrate
hydrophobicity. Notwithstanding, the corresponding substitu-
ent of the Gly substrate (an H) is too small for the S10 binding
pocket of CPD-A to show the recognition required for effective
hydrolysis by the enzyme.
According to the X-ray structures of CPD-Y13 and CPD-A,14
the active site or catalytic cavity consists of an oxyanion hole, a
C-terminal recongition site, and two hydrophobic pockets
(Scheme 3).
In CPD-Y, the hydrophobic S10 binding site consists of the
residues Thr 60, Phe 64, Glu 65, Tyr 256, Tyr 269, Leu 272,
Met 398, and disulfide 56-298, while S1 consists of Tyr 147,
Leu 178, Tyr 185, Tyr 188, Leu 245, Trp 312, Ile 340, and
Cys 341, which makes it capable of accommodating hydropho-
bic side chains at the P1 (corresponding to the N-terminal ami-
no acid moiety) and P10 (corresponding to the C-terminal ami-
no acid moiety) positions, respectively. Since P1 is fixed as
Phe, it is likely that the observed Km and Ki values are mostly
dependent upon the hydrophobicity of the C-terminal amino
acids at P10.
The X-ray structures of the S10 binding pockets of CPD-Y
and CPD-A (Fig. 3) show the rim of the S10 binding pocket
of CPD-Y to be ca. 7–10 angstroms in width and ca. 5–7 ang-
stroms in depth, while the rim of CPD-A to be ca. 10–12 ang-
stroms in width and ca. 9–11 angstroms in depth. Therefore, the
S10 binding pocket of CPD-A is larger than that of CPD-Y,
which coincides with the relation between Km (or Ki) and hy-
drophobicity, discussed above.
The hydrophobicity values being those relative to Z–Phe–
Gly, the y-intercept A of the linear regression of Fig. 1 corre-
sponds to log Km or log Ki observed or predicted for Z–Phe–
Gly, and the slope B indicates the sensitivity of substrate hydro-
phobicity towards the binding pocket in the corresponding en-
zyme (Table 2), since the exterior of the enzyme in the present
reaction system is surrounded by aqueous medium. From the
Fig. 2. Schematic illustration showing putative incorpora-
tion into S10 pocket of (a) CPD-Y and (b) CPD-A.
Scheme 3. Schematic Michaelis–Menten complex in enzymatic hydrolysis.

Y. Kanosue et al.
Bull. Chem. Soc. Jpn., 77, No. 6 (2004) 1191
Fig. 3. The S10 binding pocket of (a) CPD-Y [PDB CODE: 1YSC]13 and (b) CPD-A [PDB CODE: 5CPA]14,15 according to their
respective X-ray structures.
Table 2. The Results of Linear Regression of Fig. 1
Y = A + BX
CPD-Y
CPD-A
Dipeptides
Gly, Sar, L-Ala, L-Pro,
NMeAla
Gly, Sar, D-Ala, L-Ala,
D-Pro, L-Pro, NMeAla
Ala, Phe(p-OH),
Phe(p-OMe), Phe
Value
Error
Value
Error
Value
Error
A
B
0.509
1.62
0.032
0.07
0.476
1.52
0.097
0.23
ꢁ0:604
0.005
0.0023
0.0845
Table 3. The Residues Comprising the S10 Pocketa,bÞ
CPD-YcÞ
Log PdÞ
(octanol/water)
Thr60
ꢁ2:91
Phe64
ꢁ1:63
Tyr256
ꢁ2:42
Tyr269
ꢁ2:42
Leu272
ꢁ1:61
Met398
ꢁ1:84
CPD-AeÞ
Log PdÞ
(octanol/water)
Ile247
ꢁ1:72
Tyr248
ꢁ2:42
Glu249
ꢁ4:19
Ala250
ꢁ2:89
Ser251
ꢁ3:17
Gly252
ꢁ3:25
Gly253
ꢁ3:25
Ser254
ꢁ3:17
Ile255
ꢁ1:72
a) The residues highlighted in boldface type shows that the hydrophobicity value (log P) is more than ꢁ2. b)
Underlined residue shows the Log P value lies between ꢁ2 and ꢁ3. c) Ref. 13. d) Ref. 11. e) Ref. 15.
data of Table 2, the value B of CPD-Y is found to be about 19-
times as large as than that of CPD-A. In other words, the S10
binding pocket of CPD-Y is about 19-times more hydrophobic
than that of CPD-A.
L-Ala, D-Pro, L-Pro, NMeAla for CPD-Y and Ala, Phe(p-OH),
Phe(p-OMe), Phe for CPD-A in this case), a comparison of the
magnitude of the slope of Km plotted against hydrophobicity
might be used as a quantitative measure of the relative hydro-
phobicity of the binding pockets of different enzymes. The gen-
erality of this method awaits further experimentation on other
enzymes.
The residues comprising the S10 binding pocket of CPD-Y
and CPD-A are given in Table 3. The S10 binding pocket (con-
sisting of Phe64, Leu272, and Met398) of CPD-Y is comprised
of more hydrophobic residues than that (consisting of Ile247
and Ile255) of CPD-A. Thus, according to the structure, the
S10 binding pocket of CPD-Y is expected to be more hydropho-
bic than that of CPD-A; this assumption is consistent with a
comparison between CPD-Y and CPD-A of the slope of the lin-
ear-regression lines (vide infra). Therefore, the experimental
results presented here imply that in cases where there are sub-
strates or inhibitors that have an obvious linear relationship be-
tween their hydrophobicity and Km values (i.e., Gly, Sar, D-Ala,
Experimental
Instruments. A Shimadzu UV-240 UV–visible recording
spectrophotometer was used for kinetic measurements.
General Synthetic Method. The treatment of L-phenylalanine
(Phe) with benzyloxycarbonyl chloride (Z–Cl) and L-alanine (Ala)
with di-t-butyl dicarbonate (Boc2O) afforded Z–Phe and Boc–Ala,
respectively. Methylation of Boc–Ala with methyl iodide gave the
N-methylalanine derivative (Boc–MeAla). The reaction of ꢀ-ami-

1192 Bull. Chem. Soc. Jpn., 77, No. 6 (2004)
Hydrolysis of Dipeptides by CPD-Y and CPD-A
Calcd for C22H24N2O5: C, 66.65; H, 6.10; N, 7.06%.
no acid (Xaa = D, L-Ala, Gly, NMeAla, D, L-Pro, Aib, Sar, Phe, and
Tyr) with thionyl chloride, followed by MeOH, gave the corre-
sponding methyl ester (HCl Xaa–OMe). The coupling reactions
N-Benzyloxycarbonyl-L-phenylalanyl-sarcosine
20
(Z–Phe–
Sar). mp 61–63 ꢄC, (lit.20 46–50 ꢄC), ½ꢀꢂD ꢁ9:4, (c 0.40, EtOH),
ꢆ
20
were mediated by dicyclohexylcarbodiimide to give the corre-
sponding N- and O-protected dipeptides (Z–Phe–Xaa–OMe).
Methylation of Z–Phe–Tyr–OMe with methyl iodide gave Z–
Phe–Tyr(O–Me)–OMe. Deprotection of the C-terminus of the di-
peptides was carried out by treatment with an equivalent amount
of 1 M NaOHaq for 0.5–3 h at room temperature to give the dipep-
tide substrates, Z–Phe–Xaa.
(lit.20 ½ꢀꢂD ꢁ9:5, c 4.0, EtOH), 1H NMR (500 MHz, CDCl3) ꢁ
7.34–7.15 (10H, m), 5.91 (1H, d, J ¼ 9:0 Hz), 5.08 (1H, d, J ¼
12:5 Hz), 5.02 (1H, d, J ¼ 12:5 Hz), 4.95 (1H, q, J ¼ 8:2 Hz),
4.20 (1H, d, J ¼ 17:5 Hz), 3.94 (1H, d, J ¼ 17:5 Hz), 3.07–2.94
(2H, m), 2.88 (3H, s). Found: C, 64.71; H, 6.24; N, 7.45%. Calcd
for C20H22N2O5: C, 64.85; H, 5.99; N, 7.56%. FABMS m/z
371.1639. Calcd for C20H23N2O5 371.1608.
N-Benzyloxycarbonyl-L-phenylalanyl-2-methylalanine (Z–
20
N-Benzyloxycarbonyl-L-phenylalanyl-L-phenylalanine (Z–
20
Phe–Aib). mp 158 ꢄC, (lit.16 161 ꢄC), ½ꢀꢂD ꢁ9:7, (c 0.10,
Phe–Phe). mp 155–156 ꢄC, (lit.21 158–160 ꢄC), ½ꢀꢂD ꢁ6:89,
20
MeOH), (lit.16 ½ꢀꢂD ꢁ9:5, c 0.1, MeOH), 1H NMR (500 MHz,
(c 0.100, MeOH), 1H NMR (500 MHz, CDCl3) ꢁ 7.34–7.00
(15H, m), 6.51 (1H, d, J ¼ 6:4 Hz), 5.41 (1H, d, J ¼ 7:1 Hz),
5.05 (2H, s), 4.78–4.74 (1H, m), 4.46 (1H, m), 3.15–3.11 (1H,
m), 3.04–2.94 (3H, m). FABMS m/z 445.1766. Calcd for
C26H25N2O5 445.1765.
CDCl3) ꢁ 7.47–7.05 (10H, m), 6.48 (1H, s), 5.69 (1H, s), 5.08
(2H, s), 4.48 (1H, m), 3.05 (2H, m), 1.55 (3H, s), 1.42 (3H, s).
FABMS m/z 383.1613. Calcd for C21H23N2O5 383.1608.
N-Benzyloxycarbonyl-L-phenylalanyl-D-alanine (Z–Phe–D-
20
Ala). mp 155–156 ꢄC, ½ꢀꢂD ꢁ2:9, (c 0.10, MeOH), 1H NMR
N-Benzyloxycarbonyl-L-phenylalanyl-L-tyrosine [Z–Phe–
20
ꢄ
ꢄ
(500 MHz, CDCl3) ꢁ 7.45–7.16 (10H, m), 6.97 (1H, d, J ¼ 6:5
Hz), 6.01 (1H, d, J ¼ 8:5 Hz), 5.03 (1H, d, J ¼ 12:5 Hz), 4.95
(1H, d, J ¼ 12:5 Hz), 4.66 (1H, d, J ¼ 7:5 Hz), 4.45 (1H, q, J ¼
6:5 Hz), 3.00 (2H, d, J ¼ 6:0 Hz), 1.19 (3H, d, J ¼ 6:5 Hz).
FABMS m/z 371.1612. Calcd for C20H23N2O5 371.1608.
Phe(p-OH)]. mp 180–182 C, (lit.22 182–185 C), ½ꢀꢂD ꢁ2:56,
(c 0.100, MeOH), 1H NMR (500 MHz, CD3OD) ꢁ 7.31–7.15
(10H, m), 7.01 (2H, d, J ¼ 8:6 Hz), 6.68 (2H, d, J ¼ 8:2 Hz),
5.02 (1H, d, J ¼ 12:5 Hz), 4.96 (1H, d, J ¼ 12:8 Hz), 4.59–4.57
(1H, m), 4.38–4.35 (1H, m), 3.10–3.06 (2H, m), 2.93–2.89 (1H,
m), 2.79–2.74 (1H, m). FABMS m/z 461.1709. Calcd for
C26H25N2O6 461.1714.
N-Benzyloxycarbonyl-L-phenylalanyl-L-alanine (Z–Phe–L-
20
Ala). mp 160–162 ꢄC (lit.17 165 ꢄC), ½ꢀꢂD ꢁ10:8, (c 0.0198,
20
MeOH), (lit.17 ½ꢀꢂD ꢁ11:0, c 2, alcohol), 1H NMR (500 MHz,
N-Benzyloxycarbonyl-L-phenylalanyl-O-methyl-L-tyrosine
20
ꢄ
CDCl3) ꢁ 7.47–7.05 (10H, m), 6.47 (1H, s), 5.41 (1H, s), 5.08
(2H, s), 4.52 (1H, q, J ¼ 7:5 Hz), 4.50 (1H, t, J ¼ 7:5 Hz), 3.10
(1H, q, J ¼ 7:5 Hz), 3.06 (1H, q, J ¼ 7:5 Hz), 1.38 (3H, d, J ¼
7:5 Hz). FABMS m/z 369.1452. Calcd for C20H21N2O5 369.1450.
N-Benzyloxycarbonyl-L-phenylalanyl-glycine (Z–Phe–Gly).
[Z–Phe–Phe(p-OMe)]. mp 120–121 C, ½ꢀꢂD ꢁ4:04, (c 0.120,
1
MeOH), H NMR (500 MHz, CDCl3) ꢁ 7.38–7.17 (8H, m), 7.12
(2H, d, J ¼ 7:3 Hz), 6.97 (2H, d, J ¼ 8:2 Hz), 6.72 (2H, d, J ¼
8:5 Hz), 5.52 (2H, d, J ¼ 7:1 Hz), 5.06 (1H, d, J ¼ 12:5 Hz),
5.00 (1H, d, J ¼ 11:9 Hz), 4.65–4.61 (1H, m), 4.41–4.36 (1H,
m), 3.71 (3H, s), 3.11–3.01 (2H, m), 2.96–2.87 (2H, m). FABMS
m/z 475.1870. Calcd for C27H27N2O6 475.1892.
Enzyme CPD-A and CPD-Y. CPD-Y prepared by Oriental
Yeast Co. Ltd., and CPD-A prepared by ICN Biomedicals, Inc.,
were purchased from Wako Pure Chemical Industries, and were
used without purification.
20
mp 144–147 ꢄC, (lit.18 154 ꢄC), ½ꢀꢂD ꢁ9:9, (c 0.025, AcOH),
18
1
(lit.18 ½ꢀꢂD ꢁ10:2, c 2.73, AcOH), H NMR (500 MHz, CDCl3)
ꢁ 7.47–7.05 (10H, m), 6.52 (1H, s), 5.39 (1H, s), 5.07 (2H, s),
4.54 (1H, dd, J ¼ 6:5, 12.5 Hz), 4.06 (1H, d, J ¼ 17:9 Hz), 3.92
(1H, d, J ¼ 17:9 Hz), 3.96–3.92 (1H, m), 3.08 (2H, m). Found:
C, 63.94; H, 5.57; N, 7.92%. Calcd for C19H20N2O5: C, 64.04;
H, 5.66; N; 7.68%.
Kinetic Measurements. A spectrophotometer was used for ki-
netic measurements. Kinetics were measured by following the de-
crease in the absorbance at 230–240 nm. The reaction conditions
were kept constant over all of the kinetic measurements: tempera-
ture, 310 K; concentration of the buffer, 50 mM; and pH, 6.5.
As a typical procedure for these experiments, carboxypeptidase-
Y (4.68 mg, 7:8 ꢃ 10ꢁ8 mol) was dissolved into Tris–HCl buffer
(1 mL), and carboxypeptidase-A (1.46 mg, 4:2 ꢃ 10ꢁ8 mol) was
dissolved into Tris–HCl buffer (3 mL); the solutions were then
diluted appropriately. A stock solution of the substrate was pre-
pared as ca. 1 mM solutions by dissolving the substrate into
Tris–HCl buffer and diluting appropriately. The solution of the
substrate (3 mL) was placed in a thermostated compartment of
the spectrophotometer and incubated for 5 min. In inhibition stud-
ies, a mixture of a Z–Phe–L-Pro solution as substrate (2 mL) and a
Z–Phe–D-Xaa (Xaa = Pro or Ala) solution as inhibitor (1 mL) was
placed in a thermostated compartment of the spectrophotometer
and incubated for 5 min. After 20 mL of the enzyme solution
was added, the mixture was shaken to make it homogeneous.
The absolute absorbance of this solution was then measured. One
set of measurements was composed of data from 5 different
substrate concentrations, and the initial velocity of each substrate
concentration was calculated as the average of 3–5 runs. The di-
peptides and the hydrolysis products of dipeptide Z–Phe–Xaa by
carboxypeptidase-Y and carboxypeptidase-A were checked by
N-Benzyloxycarbonyl-L-phenylalanyl-N-methyl-L-alanine
20
(Z–Phe–NMeAla). mp 39–40 ꢄC, ½ꢀꢂD ꢁ33:2, (c 0.100, MeOH);
1H NMR (500 MHz, CDCl3) ꢁ 7.33–7.16 (10H, m), 6.23 (1H, d,
J ¼ 9:0 Hz), 5.17 (1H, q, J ¼ 7:5 Hz), 5.12 (1H, d, J ¼ 8:5 Hz),
5.09 (1H, d, J ¼ 8:5 Hz), 4.95 (1H, quintet, J ¼ 7:4 Hz), 3.09–
2.94 (2H, m), 2.86 (3H, s), 1.39 (3H, d, J ¼ 7:5 Hz). Found: C,
65.47; H, 6.46; N, 7.19%. Calcd for C21H24N2O5: C, 65.63; H,
6.25; N, 7.29%.
N-Benzyloxycarbonyl-L-phenylalanyl-D-proline (Z–Phe–D-
20
ꢄ
Pro). mp 125–126 C, ½ꢀꢂD ꢁ38:5, (c 0.102, MeOH); 1H NMR
(500 MHz, CDCl3) ꢁ 7.33–7.19 (10H, m), 5.95 (1H, d, J ¼ 8:5
Hz), 5.10 (1H, d, J ¼ 12:5 Hz), 5.02 (1H, d, J ¼ 12:5 Hz), 4.75–
4.72 (1H, m), 4.35–4.32 (1H, m), 3.54 (1H, s), 3.15–3.06 (1H,
m), 2.99–2.98 (1H, m), 2.65–2.60 (1H, m), 2.06–2.01 (1H, m),
1.86–1.73 (2H, m), 1.54–1.49 (1H, m). FABMS m/z 397.1783.
Calcd for C22H25N2O5 397.1763.
N-Benzyloxycarbonyl-L-phenylalanyl-L-proline (Z–Phe–L-
20
ꢄ
ꢄ
Pro). mp 107–108 C, (lit.19 106.5 C), ½ꢀꢂD ꢁ43:7, (c 0.130,
MeOH), 1H NMR (500 MHz, CDCl3) ꢁ 7.37–7.12 (10H, m),
5.57 (1H, d, J ¼ 9:0 Hz), 5.11 (1H, d, J ¼ 12:5 Hz), 5.06 (1H,
d, J ¼ 12:5 Hz), 4.69 (1H, dd, J ¼ 8:0, 15.1 Hz), 4.58 (1H, d, J ¼
5:5 Hz), 3.51 (1H, dd, J ¼ 8:5, 15.9 Hz), 3.06–3.03 (2H, m), 2.85–
2.80 (1H, m), 2.34–2.30 (1H, m), 2.01–1.93 (1H, m), 1.89–1.83
(1H, m), 1.78–1.71 (1H, m). Found: C, 66.64; H, 6.13; N 6.90%.

Y. Kanosue et al.
Bull. Chem. Soc. Jpn., 77, No. 6 (2004) 1193
reversed-phase HPLC; no diastereomer was detected. The mobile
phase (0.5 mL/min) consisted of acetonitrile–water (3:7) contain-
ing 60 mM AcONa buffer (pH 6.0).
J. Teague, Angew. Chem., Int. Ed., 38, 736 (1999). d) K. Yue
and K. A. Dill, Proc. Natl. Acad. Sci. U.S.A., 92, 146 (1995).
3
K. Ohkata, T. Yano, S. Kojima, Y. Hiraga, T. Yoshii, and
M. Hori, Bull. Chem. Soc. Jpn., 75, 567 (2002).
Y. Kanosue, Y. Hiraga, S. Kojima, and K. Ohkata, Chem.
Lett., 2001, 418.
Measurements of Hydrophobicity.
The free energy of
partition was derived from the following equation: ꢁꢀGret
¼
4
ꢁRT lnðKXaa=KGlyÞ¼ꢁRT½lnðVe ꢁVoÞXaa ꢁlnðVe ꢁVoÞGlyꢂ, where
KXaa=KGly is the relative partition coefficient to that of Z–Phe–
Gly, Ve is the elution volume, and Vo is the column volume in
HPLC. Analytical conditions for HPLC: all chromatographic runs
were carried out on a JASCO 980 unit equipped with a variable-
wavelength UV detector at 254 nm. The column was a 250 ꢃ 4:6
mm ODS reversed-phase column (Wakosil II 5C18HG). The mo-
bile phase (0.5 mL/min) consisted of acetonitrile–water (3:7) con-
taining 60 mM AcONa buffer (pH 6.0).
5 a) H. Zuber, Nature (London), 201, 613 (1964). b) R.
Hayashi, Y. Bai, and T. Hata, J. Biol. Chem., 250, 5221 (1975).
6 G. Jung, H. Ueno, and R. Hayashi, J. Biochem., 126, 1
(1999).
7
8
W. N. Lipscomb, Acc. Chem. Res., 22, 62 (1989).
a) W. P. Jencks, ‘‘Catalysis in Chemistry and Enzymolo-
gy,’’ McGraw-Hill, New York (1969), p. 393. b) W. Kauzman,
Adv. Protein Chem., 14, 1 (1959).
9
T. Braumann, J. Chromatogr., 373, 191 (1986).
10 A. Vailaya and C. Horvath, J. Phys. Chem. B, 101, 5875
(1997).
11 a) V. Pliska, M. Schmidt, and J. Fauchere, J. Chromatogr.,
216, 79 (1981). b) M. Charton, Prog. Phys. Org. Chem., 18, 177
(1990).
We are indebted to the Natural Science Center for Basic
Research and Development (N-BARD), Hiroshima University,
and Hiroshima Prefectural Institute of Industrial Science and
Technology for machine-time on NMR and MS instruments.
12 T. L. Hafkenscheid and E. Tomlinson, Adv. Chromatrogr.,
25, 1 (1986).
References
1
a) L. R. Pratt and A. Pohorille, Chem. Rev., 102, 2671
13 J. A. Endrizzi, K. Breddam, and S. J. Remington, Biochem-
istry, 33, 11106 (1994); PDB CODE: 1YSC.
14 D. C. Rees, M. Lewis, and W. N. Lipscomb, J. Mol. Biol.,
168, 367 (1983); PDB CODE: 5CPA.
15 S. Mangani, P. Carloni, and P. Orioli, Eur. J. Biochem., 203,
173 (1992).
16 M. Jelokhani-Niaraki, K. Yoshioka, H. Takahashi, F. Kato,
and M. Kondo, J. Chem. Soc., Perkin Trans. 2, 1992, 1187.
17 W. Grassman, E. Wunsch, and A. Riedel, Chem. Ber., 91,
455 (1958).
(2002). b) C. Hansch, Acc. Chem. Res., 2, 232 (1969). c) A. Leo,
J. Chem. Soc., Perkin Trans. 2, 1983, 825. d) F. Menger and
U. V. Venkataram, J. Am. Chem. Soc., 108, 2980 (1986). e) W.
Blokzijil and J. B. F. N. Engberts, Angew. Chem., Int. Ed. Engl.,
32, 1545 (1993). f) D. C. Rideout and R. Breslow, J. Am. Chem.
Soc., 102, 7816 (1980). g) R. Breslow, Acc. Chem. Res., 24, 159
(1991). h) R. Breslow, Green Chem., 1998, 225. i) P. A. Grieco,
Aldrichimica Acta, 24, 59 (1991). j) S. Ott and J. B. F. N. Engberts,
´
Pure Appl. Chem., 72, 1365 (2000). k) A. Lubineau, J. Auge, and
´
Y. Queneau, Synthesis, 1994, 741. l) A. Lubineau and J. Auge, Top.
18 F. Weygand and W. Steglich, Chem. Ber., 93, 2983 (1960).
19 S. Sakakibara and M. Itoh, Bull. Chem. Soc. Jpn., 40, 656
(1967).
20 C. Gerber and D. Seebach, Helv. Chim. Acta, 74, 1373
(1991).
Curr. Chem., 206, 1 (1999). m) A. Leo, C. Hansch, and D. Elkins,
Chem. Rev., 71, 525 (1971). n) C. Hansch, Acc. Chem. Res., 26,
147 (1993). o) M. H. Abraham, P. L. Grellier, and R. A. McGill,
J. Chem. Soc., Perkin Trans. 2, 1988, 339. p) M. H. Abraham,
G. S. Whiting, R. Fuchs, and E. J. Chambers, J. Chem. Soc., Perkin
Trans. 2, 1990, 291.
21 W. Konig and R. Geiger, Liebigs Ann. Chem., 727, 125
(1969).
2 a) B. R. Baker, J. Chem. Educ., 44, 610 (1967). b) S. N.
Timasheff, Acc. Chem. Res., 3, 62 (1970). c) A. M. Davis and S.
22 M. Fridkin, E. Hazum, M. Tauber-Finkelstein, and S.
Shaltiel, Arch. Biochem. Biophys., 178, 517 (1977).