Effect of enzyme-substrate interactions away from the reaction site on carboxypeptidase A catalysis

Article abstract of DOI:10.1006/bioo.1996.0026

The kinetics of 14 peptide substrates of carboxypeptidase A have been studied for the purpose of evaluating P₁-P₃/S₁-S₃ interactions. It was found that the amide group at P₁-P₂ is required

Full text of DOI:10.1006/bioo.1996.0026

BIOORGANIC CHEMISTRY 24, 290–303 (1996)
ARTICLE NO. 0026
Effect of Enzyme–Substrate Interactions Away from the
Reaction Site on Carboxypeptidase A Catalysis
J
OHN F. SEBASTIAN,1 GUIQING
LIANG, ANNISSA
J
ABARIN, KAREN
T
HOMAS AND
,
H. BONNIE
W
U
Department of Chemistry, Miami University, Oxford, Ohio 45056
E-mail: Sebastjf@miamiu.acs.muohio.edu
Received March 28, 1996
The kinetics of 14 peptide substrates of carboxypeptidase A have been studied for the
purpose of evaluating P1–P3/S1–S3 interactions. It was found that the amide group at P1–P2
is required for efficient catalysis. This observation is consistent with previously proposed
hydrogen bonding interactions, based on crystallographic data, between the P1 NH and Tyr-
248 and between the P2 carbonyl oxygen and Arg-71. In contrast, substitution of the benzamido
amide group (at P2–P3) of N-benzoylglycylglycyl-
L-phenylalanine by –CH2CH2– resulted in
more effective catalysis. In this case hydrophobic interactions are important in the ground
state and in the transition state of the rate-determining step.
1996 Academic Press, Inc.
INTRODUCTION
Bovine carboxypeptidase A (CPA) is a digestive protease that catalyzes the
hydrolysis of oligopeptides, polypeptides, and proteins at the peptide bond of C-
terminal residues with aromatic and large aliphatic side chains (1). The active site
of CPA binds the side chain and the carboxylate group of the C-terminal residue
at an apolar pocket and at Arg-145, respectively. Five binding subsites on the
enzyme, SЈ and S1 through S4 , have been reported (2). Cleavage of the peptide
1
bond occurs between PЈ and P1 of the substrate. Asn-144 and Tyr-248 also have
1
been implicated in the binding of the substrate’s terminal carboxylate group to the
enzyme. Groups believed to be involved in catalysis include Glu-270, Arg-127, and
2
ϩ
Zn . Glu-270 acts as a general base, abstracting a proton from a zinc-bound water
molecule (1). Arg-127 was shown to be a catalytic group by site-directed mutagenic
studies on rat carboxypeptidase (3). Tyr-198’s hydroxyl group appears to play a
role in catalysis by stabilizing the transition state (4). The edge-to-face arrangement
of Tyr-198 and Phe-279 constitutes a putative modifier binding site (5).
X-ray studies of bovine CPA complexed with the products of N-benzoyl-
lalanyl- -phenylalanine hydrolysis suggest that Arg-71 is hydrogen-bonded to the
amide carbonyl oxygen of the N-benzoyl- -phenylalanine product (6). The NH of
L
-pheny-
L
L
that amide is hydrogen-bonded to the hydroxyl oxygen of Tyr-248. It has been
proposed that these two hydrogen bonding interactions, away from the site of bond
1 To whom correspondence should be addressed. Fax: (513) 529–5715.
290
0045-2068/96 $18.00
Copyright 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.

EFFECT OF ENZYME–SUBSTRATE INTERACTIONS
291
breaking/bond making, occur in an intact bound substrate as well (7). There is a
hydrophobic cleft at the S1 subsite that binds apolar side chains at P1 . X-ray struc-
tures of bound phosphonates (transition state analogs) show that phenyl rings at
P2 interact favorably in an edge-to-face fashion with Tyr-248 (7). Phenyl rings at
P3 reside in the S3 subsite near the S1 cleft, and interact with Tyr-248 and Tyr-198.
In this paper, we report detailed kinetic studies of peptide substrates of bovine
CPA that probe the nature of the interactions between the P1 , P2 , and P3 substrate
sites and the corresponding sites on the enzyme and specifically test the proposed
hydrogen bonding interactions of the P1–P2 amide group with Arg-71 and Tyr-248.
MATERIALS AND METHODS
Preparation of Substrates
The reference substrate Benzoylglycyl-
L
-phenylalanine (BGP) was purchased
from Sigma and used without further purification. The range of BGP concentration
4
4
used in the kinetic study was 2.66 ϫ 10Ϫ to 9.31 ϫ 10Ϫ
M
. Glycyl-L-phenylalanyl-
L-
phenylalanine (GPP) and glycyl-
from Sigma.
L-seryl-
L-phenylalanine (GSP) were also purchased
N-(trans-Styrylacetyl)-
L
-phenylalanine (SAP) was synthesized from trans-styryl-
acetyl chloride and -phenylalanine (Sigma) as described by Haas (8). trans-Styryl-
L
acetyl chloride was synthesized from trans-styrylacetic acid (purchased from Ald-
rich) and thionyl chloride (purchased from Aldrich). About 2.7 ml of thionyl chloride
and 5 g of trans-styrylacetic acid were placed in a 120-ml flask. The flask was heated
in an 80ЊC water bath for 30 min. The black crystals of the styrylacetic acid chloride
were dissolved in 19 ml of p-dioxane. Another flask was used to suspend 3.6 g of
L-phenylalanine in 76 ml water. The suspension was stirred in an ice bath, followed
by addition of 1 N NaOH and 3.2 g sodium carbonate. The trans-styrylacetyl
chloride/dioxane solution was added to the phenylalanine solution in five portions
over a 90-min period with continued stirring in the ice bath. After the second
addition, 19 ml of diethyl ether was added. The mixture was then stirred in the ice
bath for 4 to 5 h. The product precipitated as light yellow crystals after the addition
of 1.5 ml of 85% phosphoric acid. Ethyl acetate and ether were used to wash the
crystals. The product was recrystallized from anhydrous ethanol and used without
1
further purification. The melting point was 179.2–181.0ЊC. H NMR (MeOD, 200
MHz) ͳ : 7.4–7.2 (m, 10H, phenyl rings), 6.5 (d, 1H, CH), 6.2 (m, 1H, CH), 4.7 (dd,
1H, CH), 3.2–2.9 (m, 4H, CH2). 13C NMR (MeOD, 50 MHz) ͳ : 174.6, 173.6 (carbonyl
carbons), 138.2, 134.6, 130.3, 129.4, 129.3, 128.4, 127.7, 127.2 (phenyl ring carbons),
138.4, 123.8 (UCHuCHU), 54.9 (CH), 40.8, 38.3 (CH2). MS (FAB)m/z (relative
intensity), 309 (23) Mϩ, 205 (22), 174 (20), 161 (100), 146 (54), 120 (39), 117 (91),
103 (16), 91 (72), 79 (24), 77 (15), 65 (22): calculated m/z 309.1378; measured m/
z, 309.1359. Anal. Calcd for SAP:C, 73.82; H, 6.20; N, 4.53. Found: C, 73.05; H,
6.23; N, 4.47. The range of SAP concentration used in the kinetic study was 8.54
5
4
ϫ 10Ϫ to 1.47 ϫ 10Ϫ
Benzoyl- -alanyl- -phenylalanine (BAP) was synthesized by the method de-
M.
L
L

292
SEBASTIAN ET AL.
scribed above by using
L
-alanyl- -phenylalanine (purchased from Sigma) and ben-
L
1
zoyl chloride (purchased from Sigma). The yield was 88%. H NMR (MeOD, 200
MHz) ͳ : 7.8–7.2 (m, 10H, phenyl rings), 4.6 (m, 1H, CH), 3.2 (d, 2H, CH2), 3.0 (m,
1H, CH), 1.4 (d, 3H, CH3). 13C NMR (MeOD, 200 MHz) ͳ : 174.8, 174.5, 170.0
(three carbonyl carbons), 138.3, 135.2, 132.9, 130.4, 129.5, 129.4, 128.6, 127.8 (phenyl
ring carbons), 55.1 (CH), 50.8 (CH), 38,.4 (CH2), 17.8 (CH3). MS (FAB) m/z
(relative intensity), 341 (15.3) (M ϩ H)ϩ, 190 (15.2), 176 (19.5), 166 (35.2), 148
(19.8): calculated m/z (BAP ϩ H)ϩ, 341.1496; measured m/z, 341.1513. The range
5
4
of BAP concentration used in kinetic study was 2.52 ϫ 10Ϫ to 1.26 ϫ 10Ϫ
N-(4-Phenylbutanoyl)- -phenylalanine (PBP) was prepared from 4-phenylbutyryl
chloride and -phenylalanine by the method described above. The 4-phenylbutyryl
M.
L
L
chloride was synthesized from 4-phenylbutyric acid (purchased from Sigma). The
yield was 54.6%, mp 178–182ЊC. 1H NMR (DMSO-d6 , 200 MHz) ͳ : 8.1 (d, 1H, NH)
7.3–7.1 (m, 10H, phenyl rings), 4.4 (m, 1H, CH), 3.0 (m, 2H, CH2), 2.4 (t, 2H, CH2),
2.0 (t, 2H, CH2), 1.7 (m, 2H, CH2). 13C NMR (DMSO-d6 , 200 MHz) ͳ : 173.5, 172.2
(carbonyl carbons), 141.9, 138.0, 129.2, 128.42, 128.37, 128.2, 126.5, 125.8 (phenyl
ring carbons), 53.6 (CH), 36.9 (CH2), 34.7 (CH2), 34.5 (CH2), 27.2 (CH2). MS
(MALDI)m/z (M ϩ Kϩ): calculated, 350.48; measured, 350.4. The range of PBP
5
4
concentration used in kinetic study was 6.58 ϫ 10Ϫ to 2.39 ϫ 10Ϫ
N-(4-Phenylbutanoyl)glycyl- -phenylalanine (PBGP) was prepared from 4-phe-
nylbutyryl chloride and Gly– -Phe (purchased from Sigma) as described above. 4-
M.
L
L
Phenylbutyryl chloride was synthesized from 4-phenylbutyric acid. The crude prod-
uct was recrystallized from ethanol–H2O twice. The yield was 51.1%, mp 127.0–
129.5ЊC. 1H NMR (acetone-d6 , 200 MHz) ͳ : 7.3–7.1 (m, 10H, phenyl rings), 4.7 (m,
1H, CH), 3.8 (m, 2H, CH2), 3.1 (m, 2H, CH2), 2.6 (t, 2H, CH2), 2.2 (t, 2H, CH2),
1.9 (m, 2H, CH2), 13C NMR (acetone-d6 , 200 MHz) ͳ : 173.5, 172.7, 169.8 (carbonyl
carbons), 142.9, 137.9, 130.2, 129.3, 129.1, 127.4, 126.5 (phenyl ring carbons), 54.1
(CH), 43.2 (CH2), 38.0 (CH2), 35.8 (CH2), 35.7 (CH2), 27.5 (CH2). MS (MALDI)
m/z (M ϩ Kϩ): calculated, 407.53; measured, 407.0. The range of PBGP concentra-
5
4
tion used in kinetic study was 3.44 ϫ 10Ϫ to 1.29 ϫ 10Ϫ
N-(2-Phenylacetyl)gylcyl- -phenylalanine (PAGP) was prepared from phenyl-
acetyl chloride (purchased from Aldrich) and Gly– -Phe by the procedure described
M.
L
L
above. The crude product was recrystallized from ethanol–H2O twice. The yield
1
was 61.3%, mp 135–137ЊC. H NMR (acetone-d6 , 200 MHz) ͳ : 7.3–7.2 (m, 10H,
phenyl rings), 4.7 (m, 1H, CH), 3.8 (d, 2H, CH2), 3.5 (s, 2H, CH2), 3.0 (m, 2H,
CH2). 13C NMR (acetone-d6 , 200 MHz) ͳ : 172.5, 171.5, 169.5 (carbonyl carbons),
138.0, 136.9, 130.2, 130.1, 129.2, 129.1, 127.4, 127.3 (phenyl ring carbons), 54.2 (CH),
43.4 (CH2), 43.3 (CH2), 38.1 (CH2). MS (MALDI) m/z (M ϩ Kϩ): calculated,
379.48; measured, 378.9. The range of PAGP concentration used in the kinetic
5
4
study was 3.30 ϫ 10Ϫ to 5.50 ϫ 10Ϫ
N-(3-Benzoylpropionyl)-
propionic acid (purchased from Aldrich) and
M
.
L
-phenylalanine (BPP) was synthesized from 3-benzoyl-
-phenylalanine methyl ester hydro-
L
chloride (purchased from Aldrich) in two steps by the method described by Mori-
shita et al. (9).
1. N(3-Benzoylpropionyl)- -phenylalanine methyl ester was synthesized from
L

EFFECT OF ENZYME–SUBSTRATE INTERACTIONS
293
3-benzoylpropionic acid and
L
-phenylalanine methyl ester hydrochloride. The yield
was77.8% [Lit. 69.2%(9)]. Recrystallization fromhotethanol–watertwice gavewhite
crystals. The yield was 69.8% [Lit. 60% (9)], mp 93.5–94.5ЊC [Lit 94.5–95ЊC (9)]. 1H
NMR (CDCl3 , 200 MHz) ͳ : 8.0–7.1 (m, 10H, phenyl rings), 6.2 (d, 1H, NH), 4.9 (m,
1H, CH), 3.3 (m, 2H, CH2), 3.1 (m, 2H, CH2), 3.0 (m, 2H, CH2). 13C NMR (CDCl3 ,
200 MHz) ͳ : 198.7, 172.0, 171.6 (carbonyl carbons), 136.5, 135.9, 133.3, 129.3, 128.6,
128.5,128.1,127.1(phenylringcarbons),53.2,53.3(CH,OCH3),37.9,33.8,30.0(CH2).
1
IR (KBr, Bio-Rad 3240-SPC) 3320, 1730, 1727, 1695, 1650 cmϪ .
2. N-(3-Benzoylpropionyl)- -phenylalanine was synthesized from the hydroly-
L
sis of its methyl ester. The product was recrystallized from ethyl acetate–petroleum
1
ether. The yield was 78.8% [Lit. 86.2% (9)], mp 115.5–116.0ЊC [Lit. 115ЊC (9)]. H
NMR (MeOD), 200 MHz) ͳ : 8.2 (d, 1H, NH), 8.0–7.1 (m, 10H, phenyl rings), 4.7
(m, 1H, CH), 3.3 (m, 2H, CH2), 3.1 (m, 2H, CH2), 2.9 (m, 2H, CH2). 13C NMR
(MeOD, 200 MHz) ͳ : 200.4, 174.8, 174.7 (carbonyl carbons), 138.4, 138.0, 134.3,
130.4, 129.7, 129.4, 129.1, 127.8 (phenyl ring carbons), 55.0 (CH), 38.5, 34.9, 30.5
(CH2). IR (KBr, Bio-Rad 3240-SPC); the UNHU band overlapped with the car-
1
1
boxyl UOH band at 3364 cmϪ , 1739, 1679, 1651 cmϪ . MS (FAB) m/z (relative
intensity); 326 (25) (M ϩ H)ϩ, 185 (100), 166 (40), 161 (96), 133 (16), 120 (30), 105
(18), 93 (98): calculated (BPP ϩ H)ϩ, 326.1387; measured, 326.1401. The range of
5
4
concentration of BPP used in the kinetic study was 7.89 ϫ 10Ϫ to 4.93 ϫ 10Ϫ
M.
N-(4-Benzoylbutyryl)-
L
-phenylalanine (BBP) was synthesized by the method de-
scribed by Morishita et al. (9) using 4-benzoylbutyric acid (purchased from Aldrich)
and
L-phenylalanine methyl ester hydrochloride as starting materials.
1
1. The yield for BBP methyl ester was 48.9%. H NMR (CDCl3 , 200 MHz)
ͳ : 8.0–7.1 (m, 10H, phenyl rings), 6.1 (d, 1H, NH), 4.9 (m, 1H, CH), 3.7 (s, 3H,
CH3), 3.1 (m, 2H, CH2), 3.0 (t, 2H, CH2), 2.3 (t, 2H, CH2), 2.0 (m, 2H, CH2).
13C NMR (CDCl3 , 200 MHz) ͳ : 199.7 (phUCuO), 172.1 (two carbonyl carbons
overlapped); 136.7, 135.8, 133.1, 129.2, 128.5, 128.0, 127.1 (phenyl ring carbons),
53.0 (CH), 52.3 (CH3), 37.7 (CH2), 37.2 (CH2), 35.2 (CH2), 19.9 (CH2).
2. The yield for BBP was 59.6%, mp 115–116ЊC. FT-IR (KBr); the UNHU
1
band overlapped with the carboxyl UOH band at 3290 cmϪ ; 1718, 1684, 1652, 1543
1
1
cmϪ . H NMR (CDCl3 , 200 MHz) ͳ : 7.9–7.1 (m, 10H, phenyl rings), 6.3 (d, 1H,
NH), 4.9 (m, 1H, CH), 3.2 (m, 2H, CH2), 3.1 (t, 2H, CH2), 2.3 (t, 2H, CH2), 2.0
(m, 2H, CH2); 13C-NMR (CDCl3 , 200 MHz) ͳ : 200.2 (phUCuO), 174.5, 173.3
(two carbonyl carbons); 136.6, 135.8, 133.3, 129.3, 128.6, 128.1, 127.1 (phenyl ring
carbons), 53.2 (CH), 37.3 (CH2), 37.2 (CH2), 35.2 (CH2), 20.0 (CH2). MS (MALDI)
m/z (M Ϫ Hϩ ϩ Kϩ): calculated, 378.49; measured, 378.3. The range of BBP
5
4
concentration used in the kinetic study was 3.43 ϫ 10Ϫ to 1.143 ϫ 10Ϫ
N-(3-Benzoylpropanoyl)glycyl- -phenylalanine (BPGP) was synthesized from 3-
benzoylpropionic acid, glycine methyl ester hydrochloride (purchased from Ald-
rich), and -phenylalanine methyl ester hydrochloride in four steps.
M.
L
L
1. N-(3-Benzoylpropanoyl)glycine methyl ester was prepared from 3-benzoyl-
propionic acid and glycine methyl ester hydrochloride by the method described by
1
Morishita et al. (9). The yield was 60.8%, mp 98.9–100.8ЊC. H NMR (CDCl3 , 200

294
SEBASTIAN ET AL.
MHz) ͳ : 8.0–7.4 (m, 5H, phenyl ring), 6.4 (broad, 1H, NH), 4.1 (d, 2H, CH2), 3.8
(s, 3H, CH3), 3.4 (t, 2H, CH2), 2.7 (t, 2H, CH2). 13C NMR (CDCl3 , 200 MHz)
ͳ : 198.8, 172.3, 170.4 (carbonyl carbons), 136.5, 133.3, 128.6, 128.1 (phenyl ring
carbon), 52.4 (CH3), 41.4 (CH2), 33.9 (CH2), 29.9 (CH2).
2. N-(3-Benzoylpropanoyl)glycine (BPG) was prepared via hydrolysis of its
methyl ester. The yield was 65.1%. 1H NMR (acetone-d6 , 200 MHz) ͳ : 8.1–7.5 (m,
5H, phenyl ring), 4.0 (m, 2H, CH2), 3.3 (t, 2H, CH2), 2.7 (t, 2H, CH2). 13C NMR
(acetone-d6 , 200 MHz) ͳ : 199.1, 172.8, 171.4 (carbonyl carbons), 138.0. 133.7, 129.4,
129.2, 128.7, 126.9 (phenyl ring carbons), 41.4 (CH2), 34.2 (CH2), 30.0 (CH2 , over-
lapped in solvent peaks).
3. N-(3-Benzoylpropanoyl)glycyl-L-phenylalanine methyl ester was prepared
from BPG and -phenylalanine methyl ester hydrochloride as described by Auld
L
1
and Vallee (10). The yield was 21.3%. H NMR (CDCl3 , 200 MHz) ͳ : 8.0–7.1 (m,
10H, phenyl rings), 6.7 (d, 1H, NH), 6.4 (t, 1H, NH), 4.9 (m, 1H, CH), 4.0 (m, 2H,
CH2), 3.7 (s, 3H, CH3), 3.4 (t, 2H, CH2), 3.1 (t, 2H, CH2), 2.6 (t, 2H, CH2). 13C
NMR (CDCl3 , 200 MHz) ͳ : 199.2, 172.5, 171.8, 168.7 (carbonyl carbons), 136.3,
135.9, 133.4, 129.2, 128.65, 128.57, 128.1, 127.1 (phenyl ring carbons), 53.3 (CH),
52.3 (CH3), 43.2 (CH2), 37.8 (CH2) 34.0 (CH2), 30.1 (CH2).
4. BPGP was prepared from the hydrolysis of its methyl ester as described by
Morishita et al. (9). The yield was 46.5%, mp 154.0–155.4ЊC. 1H NMR (MeOD, 200
MHz) ͳ : 7.9–7.1 (m, 10H, phenyl rings), 4.6 (m, 1H, CH), 3.7 (d, 2H, CH2), 3.3 (t,
2H, CH2), 3.1 (m, 2H, CH2), 2.5 (t, 2H, CH2). 13C NMR (MeOD, 200 MHz) ͳ : 200.1,
175.6, 174.3, 171.7 (carbonyl carbons), 138.3, 137.9, 134.5, 130.3, 129.7, 129.4, 129.2,
127.8 (phenyl ring carbons), 55.1 (CH), 43.5 (CH2), 38.4 (CH2), 34.9 (CH2), 30.7
(CH2). MS (MALDI) m/z (M ϩ Kϩ): calculated, 421.52; measured, 420.9. The
5
range of BPGP concentration used in the kinetic study was 2.64 ϫ 10Ϫ to 2.64 ϫ
4
10Ϫ
M.
N-Benzoylglycylglycyl- -phenylalanine (BGGP) was prepared by the same
L
method as described by Auld and Vallee (10). The yield was 86.5%, mp 221.4–
221.9ЊC [Lit. 218–219ЊC (10)]. FT-IR (KBr): the UNHU band overlapped with
1
1
1
the carboxyl UOH band at 3380 cmϪ ; 1745, 1662, 1631, 1552 cmϪ . H NMR
(DMSO, 200 MHz) ͳ : 8.8–7.2 (m, 10H, phenyl rings), 4.5–3 (m, CH, CH2). 13C
NMR (DMSO, 200 MHz) ͳ : 172.7, 169.2, 168.6, 166.6 (carbonyl carbons), 137.3,
133.8, 131.3, 129.90, 128.2, 128.1, 127.3, 126.4 (phenyl ring carbons), 53.5 (CH), 42.6
(CH2), 41.5 (CH2), 36.7 (CH2). MS (MALDI) m/z (M Ϫ Hϩ ϩ Kϩ): calculated,
421.49; measured, 421.4. The range of BGGP concentration used in the kinetic
5
4
study was 2.05 ϫ 10Ϫ to 2.05 ϫ 10Ϫ
M
.
Enzyme
CPAͰ (Cox) was purchased from Sigma. An aliquot was removed after the CPA
toluene suspension was vortexed. The aliquot was centrifuged at 10,000 rpm for 15
min and the supernatant was removed immediately. The CPA crystals were washed
twice with ice-cold double-distilled water and dissolved in ice-cold 3.0
M
NaCl and
diluted to 2 ml. The enzyme solution was stored at 4ЊC. The protein concentration

EFFECT OF ENZYME–SUBSTRATE INTERACTIONS
TABLE 1
295
Values of Kinetic Constants for the CPA-Catalyzed Hydrolysis of N-Acyl-
L
-phenylalanines
(kcat/KS)
4
KS ϫ 104
⌬G
ϫ 10Ϫ
⌬Gb ,b
(kcal/mol)
1
1
1
Substrate
kcat (sϪ
)
(M
)
(kcal/mol)a
(sϪ MϪ
)
O
H
N
CO2–
Ph
Ph
N
H
139 Ϯ 5
8.36 Ϯ 0.18
2.34 Ϯ 0.07
0.0
16.6
0.0
O
N-Benzoylglycyl-L-phenylalanine (BGP)
O
H
N
CO2–
Ph
Ph
5.23 Ϯ 0.18
Ϫ0.75
2.24
1.2
O
3-Benzoylpropanoyl-
L
-phenylalanine
(BPP)
H
–
CO2
Ph
N
Ph
0.709 Ϯ 0.031
0.224 Ϯ 0.025
1.37 Ϯ 0.01
2.40 Ϯ 0.02
Ϫ1.1
0.518
2.0
3.1
O
4-Phenylbutanoyl-
L
-phenylalanine (PBP)
H
–
CO2
Ph
N
Ph
Ϫ0.74
0.0933
O
N-(trans-Styrylacetyl)-L-phenylalanine
(SAP)
a Example: ⌬G ϭ RT ln(KBS PP/KBS GP) ϭ Ϫ0.75 kcal/mol.
b ⌬Gb is the binding energy of the enzyme–transition state complexes (13, 14). The apparent effect on the binding
energy of the enzyme–substrate complex in the transition state due to a change in substrate structure is calculated as
BPP
BGP
follows. (13, 14). Example: BPP/BGP: ⌬Gb ϭ ϪRT ln͕[k /KBS PP]/[k
/KBS GP]͖ ϭ 1.2 kcal/mol.
cat
cat
was determined spectrophotometrically. The molar absorptivity of CPA at 278 nm
1
1
is 6.41 ϫ 104 MϪ cmϪ (11). The protein concentration of CPA stock solution is
4
about 2 ϫ 10Ϫ
M.
Buffer Solution
A 1.0
M
NaCl/0.05
M
Tris : pH 7.50 buffer at 25.0ЊC was used for the kinetic assay.
The pH was measured with a Beckman Expandomatic pH meter. All solutions
were stored at 4ЊC and allowed to warm to room temperature before use.
Kinetic Measurements
Kinetic data were collected on a Cary E1 spectrophotometer equipped with a
thermostated cell compartment. The cell compartment was maintained at 25.0 Ϯ
0.1ЊC with a water bath circulator.

296
SEBASTIAN ET AL.
TABLE 2
Values of Kinetic Constants for the CPA-Catalyzed Hydrolysis of N-Benzoylglycylglycyl-
phenylalanine and Two Derivatives
L-
(kcat/KS)
4
KS ϫ 104
⌬Ga
(kcal/mol)
ϫ 10Ϫ
⌬Gb ,a
(kcal/mol)
1
1
1
Substrate
kcat (sϪ
)
(M
)
(sϪ MϪ
)
O
H
N
H
N
CO2–
Ph
Ph
N
H
18.8 Ϯ 0.8b
7.03 Ϯ 0.08b
3.98 Ϯ 0.06
0.739 Ϯ 0.019
Ϫ0.10
Ϫ0.44
Ϫ1.4
2.67
1.1
Ϫ0.4
Ϫ1.4
O
O
N-Benzoylglycylglycyl-L-phenylalanine
(BGGP)
O
H
CO2–
Ph
N
N
H
O
141 Ϯ 7
35.4
O
Ph
N-(3-Benzoylpropanoyl)glycyl-L-
phenylalanine (BPGP)
O
H
CO2–
Ph
Ph
N
N
H
O
134 Ϯ 3
181
N-(4-Phenylbutanoyl)gylcyl-L-
phenylalanine (PBGP)
a Defined in Table 1; reference substrate is BGP.
b In good agreement with previously reported values (17).
Data Analysis
The kinetic parameters KM and Vmax were obtained from a nonlinear least-squares
fit of the Michaelis–Menten equation. For inhibition experiments, the form of the
inhibition and Ki values were determined as described by Uhr et al. (12). The errors
given are standard errors.
Molecular Modeling
Molecular models were generated from Protein Data Bank (PDB) files via CS
Chem3D Pro, version 3.2.
RESULTS
The N-acyldipeptide N-benzoylglycyl- -phenylalanine is the reference substrate
L
in this study. According to the crystallographic studies of a variety of transition-
state analogs, the P1 NH of the benzamido group of this substrate in the transition-
state complex should be hydrogen-bonded to the hydroxyl oxygen of Tyr-248 and
the P2 carbonyl oxygen of the amide hydrogen-bonded to Arg-71 (7). We have

EFFECT OF ENZYME–SUBSTRATE INTERACTIONS
297
TABLE 3
Values of Kinetic Constants for the CPA-Catalyzed Hydrolysis of Various N-Acyldipeptides
(kcat/KS)
4
⌬Ga
(kcal/mol)
ϫ 10Ϫ
⌬Gb ,a
(kcal/mol)
1
1
1
Substrate
kcat (sϪ
)
KS ϫ 104
(
M
)
(sϪ MϪ
)
O
H
–
CO2
Ph
N
H3C
N
H
35b
49.1 Ϯ 2.9b
1.0
0.0
0.71
1.9
O
N-Acetylglycyl-
L
-phenylalanine (AcGP)
O
H
CO2–
N
Ph
N
H
139 Ϯ 5
8.36 Ϯ 0.18
2.37 Ϯ 0.02
16.6
0.0
0.3
O
Ph
N-Benzoylglycyl-
L-phenylalanine (BGP)
O
H
CO2–
N
Ph
N
H
24.0 Ϯ 0.5
Ϫ0.75
Ϫ0.14
Ϫ1.4
10.1
52.5
O
Ph
N-(2-Phenylacetyl)glycyl-L-phenyl-
alanine (PAGP)
O
H
N
CO2–
Ph
Ph
N
H
347b
6.6 Ϯ 1.0b
Ϫ0.7
Ϫ1.4
O
N-Hydrocinnamolyglycyl-
alanine (HCinGP)
L
-phenyl-
O
H
N
–
Ph
CO2
Ph
N
H
134 Ϯ 3
0.739 Ϯ 0.019
181
O
N-(4-Phenylbutanoyl)glycyl-L-phenyl-
alanine (PBGP)
a Defined in Table 1.
b Values taken from Ref. (18).
tested this notion by measuring the kinetics of the CPA-catalyzed hydrolyses of N-
3-benzoylpropanoyl- -phenylalanine, N-4-phenylbutanoyl- -phenylalanine, and N-
(trans-styrylacetyl)- -phenylalanine and comparing the results with those for BGP.
L
L
L
The results are given in Table 1. KS values are listed as it is known that KM Ȃ KS
for the hydrolysis of peptide substrates catalyzed by bovine CPA at 25ЊC (15).
Substitution of the benzamido P1 NH by CH2 (giving BPP) causes nearly an eightfold
reduction in kcat/KS . BPP is a sufficiently poor substrate that it could be tested as
a modifier of BGP hydrolysis and was found to be a competitive inhibitor with
4
Ki ϭ 1.25 Ϯ 0.20 ϫ 10Ϫ
M, in reasonably good agreement with the KS value for
BPP. It has been established previously that 2,2-dimethyl-2-silapentane-5-sulfonate
(DSS) activates the CPA-catalyzed hydrolysis of BGP (16). With BPP as substrate,

298
SEBASTIAN ET AL.
TABLE 4
Values of Kinetic Constants for the CPA-Catalyzed Hydrolysis of Tripeptides and N-Benzoyl-L-
alanyl-L-phenylalanine
(kcat/KS)
4
KS ϫ 104
⌬Ga
(kcal/mol)
ϫ 10Ϫ
⌬Gb ,b
(kcal/mol)
1
1
1
Substrate
kcat (sϪ
)
(
M
)
(sϪ MϪ
)
Ph
O
+
H
N
–
H3N
CO2
Ph
N
231 Ϯ 5
0.695 Ϯ 0.015
Ϫ1.5
332
Ϫ1.8
H
O
Glycyl-L-phenylalanyl-L-phenylalanine
(GPP)
O
N
H
N
CO2–
Ph
Ph
H
O
271 Ϯ 20
0.818 Ϯ 0.068
Ϫ1.4
331
Ϫ1.8
N-Benzoyl-
L
-alanyl-
L
-phenylalanine
(BAP)
HO
O
+
H
–
H3N
CO2
Ph
N
12.7 Ϯ 2.1
1.66 Ϯ 0.07
Ϫ0.096
7.6
0.5
1.8
N
H
O
Glycyl-
L
-seryl-
L
-phenylalanine (GSP)
O
+
H
–
H3N
CO2
Ph
N
N
H
27.5b
34.3b
0.84
0.80
O
Glycylgylcyl-L
-phenylalanine (GGP)b
a Defined in Table 1; reference substrate is BGP.
b Data taken from the Ph.D. dissertation of Roger Poorman, 1979, p. 51, Miami University.
2
DSS is a competitive inhibitor with a Ki ϭ 6.21 Ϯ 1.16 ϫ 10Ϫ
M
. Replacement of
the ketonic CuO group of BPP by CH2 (yielding PBP) further diminishes kcat/KS
by a factor of 4. The poorest substrate in this study is SAP, where the CuC group
is employed to model the partial double-bond character of the benzamido carbonyl
carbon–nitrogen bond. SAP is an uncompetitive inhibitor of BGP hydrolysis with
5
Ki ϭ 5.78 Ϯ 1.50 ϫ 10Ϫ
M. This result indicates that SAP binds four times [KS (for
SAP/Ki] more strongly to the CPA и BGP complex than to free CPA.
An approach similar to that employed to study the N-acyldipeptides was carried
out with the N-acyltripeptides listed in Table 2. The P3–P2 site of BGGP is examined
here. In marked contrast to the dipeptide situation, substitution of the P2 NH of
the benzamido group of BGGP by CH2 to give BPGP results in a 13-fold increase
in kcat/KS . Substitution of the ketonic carbonyl (corresponding to the P3 carbonyl)
of BPGP by methylene to produce PBGP provides an additional 5-fold increase in
kcat/KS .

EFFECT OF ENZYME–SUBSTRATE INTERACTIONS
299
F
IG. 1. P1Ј–P3 regions of Cbz-Phe–ValP–(O)Phe [ZFVP(O)F]. ZFVP(O)F is O-[[(1R)-[[N-(phenylmeth-
oxycarbonyl)- -phenylalanyl]amino]isobutyl]hydroxyphosphinyl]- -3-phenyllactate.
L
L
The kinetics of five substrates (Table 3) were determined for the purpose of
examining the effect of increasing apolar character of the N-acyl group of N-
acyldipeptides on the kinetic parameters. In general, as the size of the apolar moiety
increases, so does the kcat/KS value.
The P1/S1 interaction was further probed by the four substrates listed in Table
4. Substitution for hydrogen at the P1 site interacts kcat/KS . The data show that
the apolar benzyl group is significantly more effective than the smaller but polar
hydroxymethyl moiety and that N-acyldipeptides can be quite specific substrates if
there is a side-chain group other than hydrogen at P1 (6).
DISCUSSION
Crystallographic studies of three tripeptide phosphonates, a dipeptide ketomethy-
lene, and the three C-terminal residues of the potato inhibitor have indicated a
common mode of binding to CPA. A hallmark of this normal binding mode is a

300
SEBASTIAN ET AL.
F
IG. 2. (A) Active site with ZFVP(O)F (7). (B) Active site, inhibitor deleted.
hydrogen bonding interaction between the P1 NH and the Tyr-248 hydroxyl and a
hydrogen bond between the P2 carbonyl oxygen and Arg-71. The PЈ–P3 regions of
1
the normal binding inhibitor Cbz-Phe–ValP–(O)Phe (O-[[1R)-[[N-(phenylmeth-
oxycarbonyl)-
L
-phenylalanyl] amino]isobutyl]hydroxyphosphinyl]- -3-phenyllace-
L
tate) are indicated in Fig. 1 (7). The active-site region of CPA with and without
the bound inhibitor is shown in Fig. 2 (7). It has been proposed that this mode also
obtains for productive binding of polypeptide substrates of CPA (7). X-ray studies
cast considerable doubt that dipeptides with a glycyl P1 and their analogs possess
these interactions. For example, the N-terminal benzamido portion of the ketone
5-benzamido-2-benzyl-4-oxopentanoic acid, an analog of BGP, was partially disor-
dered in the crystal structure and as a consequence there was no direct evidence
of the hydrogen bonding interactions expected for normal binding (19). However,
the rather large differences in kcat/KS found for BGP, BPP, and PBP (Table 1) [32-
fold for (kcat/KS)BGP/(kcat/KS)PBP] implicate such interactions in the transition state
for BGP hydrolysis; BGP is bound 1.2 kcal/mol (compare ⌬Gb values in table 1)
more strongly than BPP and 2.0 kcal/mol more tightly than PBP in the transition
state; however, BPP and PBP bind more tightly than BGP in the ground state
(Table 1). This is likely a result of favorable hydrophobic interactions of the less

EFFECT OF ENZYME–SUBSTRATE INTERACTIONS
301
F
IG. 3. Dependence of ln(kcat/KS) on ȏ. Substrates (RCO–Gly–L–
L-Phe): ᭜, CH3 ; ᭡, Ph; ᭹, PhCH2 ;
᭿, PhCH2CH2 ; ᭝, PhCH2CH2CH2 .
polar BPP and PBP with the active site and poorer solvation of these two substrates
by solvent water. BPP and PBP are less polar than BGP because BPP is a ketone
and the polar benzamido group of BGP has been changed to the apolar PhCH2CH2
moiety in PBP. BPP is a competitive inhibitor of BGP hydrolysis, indicating that
both bind to the same active-site region. DSS, an activator of BGP hydrolysis, is
a competitive inhibitor of BPP hydrolysis. These data suggest that while BGP and
BPP bind at the same active-site region, they differ in the precise details of that
binding. Possibly the 3-benzoylpropanoyl moiety of BPP binds at least partially to
the S1 hydrophobic cleft. Finally, it should be noted that the kcat/KS for BGP is
nearly 180 times that of SAP, the poorest substrate in these studies. The rigidity
of the CuC bond and the lack of the hydrogen bonding interactions that are
available to BGP apparently prevent tight binding of SAP in the transition state
[⌬Gb for SAP is 3.1 kcal/mol higher than that for BGP (Table 1)].

302
SEBASTIAN ET AL.
F
IG. 4. Bar plots of the values of ⌬G, ⌬Gb , and kcat for the substrates of CPA. The data were from
Table 1 (A), Table 2 plus BGP (B), Table 3 (C), and Table 4 plus BGP (D). The hatched bars indicate
the values of ⌬G; the white bars indicate the values of ⌬Gb ; and the black bars indicate the values of
kcat . The error bars are standard errors.
With the tripeptide substrate BGGP (Table 2), successive replacement of the
benzamido NH and CuO groups by CH2 (to give BPGP and PBGP, respectively)
increases kcat/KS markedly. Ground-state binding as measured by KS is also en-
hanced. The flexibility of the 3-benzoylpropanoyl group of BPGP and, to a greater
extent, of the 4-phenylbutanoyl group of PBGP apparently allows for favorable
apolar interactions with the S2–S3 subsite in the ground state and in the transition
state. The five N-acyldipeptides listed in Table 3 illustrate the importance of hy-
drophobic effects in CPA catalysis. Plots of kcat/KS against Hansch’s ȏ parameter
(20, 21) (a measure of hydrophobic character) for each of these substrates are
shown in Fig. 3 and clearly demonstrate the significance of hydrophobic interactions
at P2/S2 in the transition state.
The ⌬G, ⌬Gb , and kcat data for the substrates are graphically depicted in Fig. 4.
Relative to BGP, the structural changes required to form BPGP and PBGP (Fig.
4B) stabilize the ground state and the transition state equally; hence, the kcat values
for the three substrates are essentially the same. The transition states for HCinGP,
GPP, and BAP are stabilized more than the ground states (relative to BGP) (Figs.
4C and B), and thus these three substrates have kcat values greater than that for
BGP. For BPP, PBP, SAP, BGGP, PAGP, and GSP (Figs. 4A–D), the transition
states are destabilized more than the ground states are stabilized, resulting in smaller
kcat values relative to BGP. Note that the benzamido amide group of BGGP slightly

EFFECT OF ENZYME–SUBSTRATE INTERACTIONS
303
stabilizes the ground state but significantly destabilizes the transition state. Both
the ground state and transition state are destabilized for AcGP and GGP; this also
results in smaller kcat values for these substrates.
ACKNOWLEDGMENTS
The Cary E1 spectrometer used in these studies was purchased with funds provided in part by the
National Science Foundation (NSF BIR-9120520) for which we are grateful. We thank Dr. Carolyn
Cassady for help with the MS measurements.
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