J. Biol. Macromol., 14(1), 27-41 (2014)

Regular article
Amino acid substitution reveals the role of V-shape helix on construction of yeast
carboxypeptidase Y
Mai Makino1, Takehiko Sahara2, Naoki Morita3, and Hiroshi Ueno1
1

Laboratory of Applied Microbiology and Biochemistry,
Nara Women’s University, Kitauoyanishi-machi,
Nara 630-8506, Japan2
Bio-Design Research Group, Bioproduction Research Institute,
National Institute of Advanced Industrial Science and Technology,
Central 6, 1-1-1 Higashi, Tsukuba,
Ibaraki 305-8566, Japan3
Molecular and Biological Technology Research Group, Bioproduction Research Institute,
National Institute of Advanced Industrial Science and Technology,
2-17-2-1, Tsukisamu-Higashi, Toyohira-ku, Sapporo,
Hokkaido 062-8517, Japan

Received January 28, 2014; Accepted February 4, 2014

Carboxypeptidase Y (CPY) is a vacuolar serine-type protease of yeast Saccharomyces
cerevisiae to release wide variety of L-amino acid including proline from C termini of
peptides and proteins.

According to three-dimensional structure of mature CPY

determined crystallographically, there is a characteristic region constructed with two
adjacent α-helices, called ‘V-shape helix’, which lies over the active site cavity just like adoor.

In order to clarify the role of V-shape helix, we attempted to immobilize the V-shape

helix by introducing amino acid substitution to create additional disulfide bridge between
the V-shape helix and the main body of CPY. Resulting mutant enzymes presented with
misfolding or hydrolase activity loss, suggesting that replacement of the residues at or near
V-shape helix would influence on the protein folding and enzyme activity. The results ofthis report demonstrated that the insertion domain including V-shape helix in α/β hydrolasefold serine carboxypeptidases plays a role in the construction of functional enzyme.
Key words: carboxypeptidase, serine protease, α/β hydrolase fold, protein folding, substrate
recognition

Introduction
To whom correspondence should be
addressed. Fax: +81-742-20-3448;
E-mail address: jam_makino@cc.narawu.ac.jp

Carboxypeptidase Y (CPY) is a vacuolar
serine-type protease of yeast Saccharomyces

28

V-shape helix of yeast carboxypeptidase Y

cerevisiae to hydrolyze esters, amides, anddisulfide bonds, Cys193-Cys207 and Cys262-

anilides, not only to release wide variety of L-

Cys268, are located at the beginning andamino acid including proline from C termini

ending of the V-shape helix, just like hinges.

of peptides and proteins (1-4).

The matureThus, it is projected that V-shape helix may

form of CPY exists as a 61-kDa monomer

move like a door during the catalysis. Most

including 421 amino acid residues.

Three-

lipases exhibit an α/β hydrolase fold and also

dimensional structure of mature CPY washave characteristic structural feature, which isdetermined by X-ray crystallography (5). It

constructed with a mobile helical fragment,is suggested that the structure belongs to a

named lid or flap, and this part participates ingroup of α/β hydrolase-fold family which

controlling the access of substrate to the active

includes serine carboxypeptidase II fromsite

wheat (CPWII) and lipase from Geotrichum

carboxypeptidases, the motion of V-shape

The conformation of α/β

candidum (6-8).

(13-17).

However,inthehelix has not been discussed so far.

hydrolase fold proteins can be divided into

CPY utilizes C-terminal carboxylate andtwo domains: core domain that is generallyside chains of the substrates for the substrate

similar among the fold family and insertion

recognition where a hydrogen bond network

domain that is composed of diverse structureand substrate binging sites, S1’ and S1~S5

elements.

subsites, respectively, play critical roles (5,18-

The residues 180~317 of CPY

correspond to the insertion domain.

24).

Interestingly, a part of V-shape helix

CPY has a characteristic region (residues

constru cts the S3 and S5 subsites. Hence, it

204~251) constructed with two adjacent α-

is reasonable to assume that V-shape ishelices, called ‘V-shape helix’, that is a part ofinvolved

the insertion domain (Fig. 1) (5).

mechanism by approaching to the active site

helix

is

commonV-shapeamongserineinthesubstrate

recognition

cavity.

α/βIn this study, it is of our aim to clarify thehydrolase fold to include CPY, CPWII, androle of V-shape helix in the construction of

human

CPY.

carboxypeptidases,

protective

thoseprotein/havingcathepsinA

In order to verify the involvement of(PPCA), where CPY has the largest V-shape

V-shape helix in the substrate accessing, we

helix (6,9).

According to the crystal

attempted to immobilize the V-shape helix bystructure of mature CPY, V-shape helix lies

introducing amino acid substitutions, so thatover the active site cavity in which theadditional disulfide bridge between the V-

essential catalytic triad, Ser146, His397, andshape helix and the core domain of CPY

Asp338, are positioned (5,10-12).

would be created.

Two

Two pairs of nearest

M. Makino, et al.

29

residues, Tyr225 and Pro315, and Gln228 andLys314, were selected for the mutation sites
based upon the distance of Cβ atoms between
the residues on V-shape helix and that on core
domain, where distances for Tyr225-Pro315
and Gln228-Lys314 are 4.5 Å and 5.8 Å,
respectively (Fig. 2).

All these residues

seem to be unrelated to the substrate accessing
andrecognition.

Doublemutants,

Y225C/P315C and Q228C/K314C, and single
mutants, Y225C, P315C, Q228C, and K314C
were produced by using a novel yeast
expression system.

Also constructed werethe mutants, Y225A, Y225F, P315S, andP315G, in order to verify the effects of themutation at or near V-shape helix on protein
expression and enzyme activity of CPY.

Fig. 1 Three dimensional structure of
mature CPY determined by X-ray
crystallography (PDB ID: 1YSC)
V-shape helix (Ser204~Thr251) iscolored in green. The catalytic triad(Ser146, His397, and Asp338) are
shown in red stick. Disulfide bonds
are shown in orange thick stick.

Materials
S.

Materials and methods

his3Δ1,

cerevisiae
leu2Δ0,

BY4741prc1Δ(MATa,

met15Δ0,

ura3Δ0,

prc1Δ::kanMX4) was obtained from Thermo

Fig. 2 Position of residues replaced by Cys
Two pairs of residues (Tyr225 and Pro315 (A), and Gln228 and Lys314 (B)) wereselected for replacement by Cys. The distance of Cβ atoms of Tyr225 and Pro315
was 4.5 Å and that of Gln228 and Lys314 was 5.8 Å. V-shape helix was colored ingreen.

30

V-shape helix of yeast carboxypeptidase Y

Fisher Scientific (Waltham, MA, USA).

Nakarai Tesque (Kyoto, Japan).

Cloning vector pGEM-T Easy Vector waspurchased from Promega (Madison, WI,

PlasmidUSA).

mutagenesis

QuikChange

Site-Directed

Mutagenesis Kit was purchased from Agilent

constructions

andsite-directed

The sequence of CPY was obtained fromTechnologies (Santa Clara, CA, USA).

Saccharomyces

genomedatabase

Restriction enzymes used in this study were(http://www.yeastgenome.org/).

Molecularpurchased from Takara Bio (Shiga, Japan),modeling of mature CPY was carried out with

Toyobo (Osaka, Japan), and Roche (Basel,

Discovery Studio 2.5.5 (Accelrys, San Diego,

Switzerland). T4 DNA ligase was obtained

CA, USA) using crystal structure of maturefrom Takara Bio. Blend Taq and Taq DNA

CPY (PDB ID: 1YSC).

Polymerase were purchased from Toyobo andextracted from Saccharomyces cerevisiae

New England Biolabs (Ipswich, MA, USA),wild-type strain using potassium acetate

respectively.

TOYOPEARL Butyl-650M

method (25).

resinhydrophobic

interaction

length wild-type CPY was amplified fromchromatography was obtained from Tosoh

genomic DNA by PCR with Blend Taq DNA(Tokyo, Japan).

Quick Start Bradford

polymerase and oligonucleotide primer pair,Protein Assay Kit was purchased from Bio-

SmaI-prc1-F and prc1-XhoI-R (Table 1). In

Rad (Hercules, CA, USA).

N-benzoyl-L-

this PCR, SmaI restriction site and XhoI

tyrosine p-nitroanilide (BTpNA) was obtained

restriction site were introduced to the 5’- andfrom Sigma-Aldrich (St. Louis, MO, USA).

3’- termini of prc1, respectively.

for

Genomic DNA was

Prc1 fragment encoding full-

The PCR

Benzyloxycarbonyl-L-phenilalanyl-L-leucine(Z-Phe-Leu-OH) and benzyloxycarbonyl-Lphenilalanyl-L-proline (Z-Phe-Pro-OH) wereobtainedfrom

Bachem(Bubendorf,

Switzerland), and N-acetyl-L-tyrosine ethyl
ester (Ac-Tyr-OEt) was obtained from Peptide
Institute, Inc. (Osaka, Japan).

Synthetic

oligonucleotide primers were purchased fromJapan Bio Services (Saitama, Japan) andOperon Biotechnologies (Tokyo, Japan).
Other chemicals were purchased from Wako
Pure Chemical Industries (Osaka, Japan) and

Fig. 3 Schematic diagram of theconstructed plasmid vector
pLTCV5HGWT

M. Makino, et al.

31

product was inserted into pGEM-T easy

oligonucleotide primer pair as shown in Table

vector and constructed pGCWT.

Then

1. Insertion of prc1 fragment to the plasmid

pGCWT was digested with SmaI and XhoI andvector and each mutation introduced into thethe fragment was ligated into pLTex321sV5Htargetthat was digested with SmaI and XhoI.

sequencing entrusted to Fasmac (Kanagawa,ResultingJapan).

recombinant

CPY

expression

sites

were

confirmedbyDNA

plasmid vector pLTCV5HGWT, a novel yeast
low-temperature inducible expression vector,

Expression and purification of recombinant

was thus constructed (Fig. 3).

wild-type and mutant CPYs

Plasmid vectors for CPY variants were

Tyr225 and Pro315 substituted mutant

constructed by using a QuikChange Site-

proteins were expressed and purified by the

Directed Mutagenesis method employing

same method for the recombinant wild-type

pLTCV5HGWT as the template and each

CPY preparation as follows: Saccharomyces

Table 1 Oligonucleotide primers using for construction of plasmid vectors of wildtype CPY, Cys-introduced mutants (Y225C, P315C, Y225C/P315C, Q228C, K314C,
and Q228C/K314C) and Tyr225- and Pro315-substituted mutants (Y225A, Y225F,
P315S, and P315G) by site-directed mutagenesis method. SmaI-prc1-F and prc1XhoI-R were used for amplifying prc1 fragment. SmaI restriction site and XhoI
restriction site are underlined, respectively. The other primers were used as forward
primers for DNA mutagenesis. The sequence of each forward primer is only shown.
Mutation sites are indicated by double-underlines.

32

V-shape helix of yeast carboxypeptidase Y

cerevisiae BY4741prc1Δ (MATa, his3Δ1,

ml/g wet weight of 50 mM sodium acetate

leu2Δ0, met15Δ0, ura3Δ0, prc1Δ::kanMX4)

buffer, pH 5.0, and adjusted to pH 5.0 and

was transformed with expression vector using

incubated at room temperature for 18 h tolithium

activate precursor CPY.

acetatemethod(26).

The

The activatedtransformant was selected on SD-ura plate

solution was dialyzed against 10 mM sodium

medium, contained 0.67% Yeast Nitrogen

phosphate buffer, pH 7.0.

Base without Amino Acids, 2% glucose, 2%

Activated CPY was purified from theagar, 30 µg/ml L-leucine, 20 µg/ml

dialyzed solution by hydrophobic interaction

histidine monohydrochloride monohydrate,

chromatography (HIC) on TOYOPEARL

30 µg/ml adenine hemisulfate dehydrate, andButyl-650M.

20 µg/ml L-methionine. The selected yeast

with 10 mM sodium phosphate buffer, pH 7.0,

cell was grown in YPD medium, contained

containing 2 M ammonium sulfate and applied

2% polypeptone, 1% yeast extract, and 2%

to a column of TOYOPEARL Butyl-650M

glucose at 28 °C until OD600 reached over 1.0.

(1.5 × 5 cm) equilibrated with the same buffer.

The temperature was lowered at 20 °C and the

The column was washed with 10 mM sodium

culture was shaken for 3 days in order tophosphate buffer (pH 7.0) containing 0.1 M

induce recombinant wild-type CPY or mutant

ammonium sulfate and then the eluate with 10

protein expression. The cells were harvestedmM sodium phosphate buffer (pH 7.0) wasby centrifugation at 2,000 ×g.

collected.

Purification of each recombinant CPY was

The

The solution was diluted 1:5

eluatewasconcentrated

byperformed according to the method described

ultrafiltration and treated at 50 °C for 10 min.

previously (27).

After centrifugation at 10,000 ×g, theThe cell pellet was mixed

with 0.2 ml/g wet cell of chloroform andstirred at room temperature for 30 min.

supernatant was pooled.

Then

0.4 ml/g wet cell of H2O was added and theProtein concentration

pH of the mixture was adjusted to 7.0 and wasProtein concentration in each enzyme

kept stirred at room temperature for 2 h. The

solution was determined by Quick Start

pH was readjusted to 7.0 and the mixture was

Bradford Protein Assay Kit (Bio-Rad) using

left at room temperature for 16 h under stirring.

bovine serum albumin as a standard protein.

The lysate was centrifuged at 5,000 ×g andammonium sulfate fractionation was carriedout for the supernatant first at 20% then at
90%.

The precipitates were dissolved in 3

SDS-PAGE and Western blotting
The expression of recombinant CPYs andprotein purity of each purification step wereM. Makino, et al.

33

evaluated by SDS-PAGE and Western blotting.

Technologies, Tokyo, Japan).

Polyacrylamide gel at 10% containing 0.1%

activities of those enzymes were measured by

SDS was used for SDS-PAGE and the gel wasamino acid analysis using LaChromUltra U-

stained with

HPLC system (Hitachi High-Technologies)

0.1% (w/v)

CBB R-250

PeptidaseIn

for the increase of L-leucine or L-proline atWestern blotting, the proteins within the SDS-

pH7.0, products of the hydrolyses of Z-Phe-

PAGE gel

Leu-OH or Z-Phe-Pro-OH, respectively, at

contained dye after electrophoresis.

were transferred

to

PVDF

membrane electrically with Trans-Blot SD

room temperature.

Semi-Dry Electrophoretic Transfer Cell (Bio-andRad) with the use of CAPS buffer, pH 11.

spectrophotometric

After blocking with skim milk at room

Hydrolysis of Ac-Tyr-OEt at pH 8.0 wastemperature for 1 h or at 4 °C overnight, themeasured at 237 nm at room temperature.

membranerabbitKinetic parameters, kcat and Km, werepolyclonal anti-CPY antiserum (1:10,000)

calculated from Michaelis-Menten equation

prepared in our laboratory and horseradish

and Hanes-Woolf plots.

wasincubatedwithperoxidase-conjugatedanti-rabbit

IgG

secondary(1:100,000)(GE

antibodies

Healthcare, Buckinghamshire, UK) in turn to

P315G

were

Esterase activities of WT
determined

by

the

method(27,29):Results

detected mature wild-type CPY and mutant
proteins.

The immuno-positive bands werevisualized with ImmunoStar LD (Wako) andwere exposed on an instant film using ECL
Mini-camera (GE Healthcare).

Expression and purification of recombinant
wild-type and mutant CPYs
The constructed plasmid, pLTCV5HGWT,
was introduced into a S. cerevisiae strain
BY4741prc1Δ and the transformant was

Kinetic analyses of hydrolysis activities ofcultured in YPD medium.

WT and mutant enzymes

was expressed inducibly by lowering the

Wild-type CPY

Anilidase activities of wild-type CPY andculture temperature from 28 to 20 °C. Wet

soluble mutant enzymes toward BTpNA werecell paste of 11.2 g was obtained from 1 ldetermined spectrophotometrically (27,28).

culture.

The release of p-nitroaniline from BTpNA at

produced as an inactive precursor in the cells,

pH 7.0 was monitored at 410 nm at room

they were extracted from the cells and weretemperature

U-2000A

activated by the conventional method (27).

High-

After the activation, mature wild-type CPY

Spectrophotometer

using(HitachiSince the recombinant CPY was

34

V-shape helix of yeast carboxypeptidase Y

was detected on SDS-PAGE at approximately

hence, and V-shape helix plays a role in

61 kDa.

After the heat treatment, WT was

correcting folding process because Tyr225 isyieldedwithone of the component residues of V-shape

approximately

100-foldhelix. Among the mutants on Pro315, only

purification (Fig. 4 and Table 2).
Expression vectors for CPY variants were

P315C aggregated, while the other mutants,

constructed by site-directed mutagenesis

P315S and P315G, were purified as soluble

using pLTCV5HGWT as a template.

proteins. Thus, Pro315 may not be involvedDNA

sequence analysis confirmed the codonchanges. Each mutant protein was expressedin the transformed yeast cell and purified by
the same method as WT preparation.
Western blot analysis of the cell suspension of
the transformants after inductive cultivation
showed that all of cysteine introduced mutants
were expressed in the yeast cells, respectively.
However, Y225C, P315C, and double mutants,
Y225C/P315C

andQ228C/K314C,

precipitated during the purification steps,
while the other mutants, Q228C, and K314C,
were recovered in soluble fractions and were
purified (Fig. 5(A)).

This result suggeststhat the precipitated mutant proteins tended to
misfold easily.

Both double mutants

aggregated, suggesting that the excessive
presence of cysteine at and around V-shape
helix altered the protein folding.
The result that Q228C and K314C were
purified as soluble proteins implies that
substitution of Gln228 and Lys314 may notaffect the protein folding. On the other hand,all of Tyr225 substituted mutants aggregated(Fig. 5(B)).

This result suggests thattyrosine at 225 may be important for folding;Fig. 4 SDS-PAGE (A) and Western
blotting (B) of recombinant wild-type
CPY at purification steps
Yeast cells in which recombinant WT is
expressed were lysed by chloroform andthe supernatant was treated withammonium sulfate. Pro-CPY in the
precipitates was activated at pH 5.0 andactivated mature CPY was purified byhydrophobic
interaction
chromatography on TOYOPEARL
Butyl-650M and heat treatment as
described in the text.
In Western
blotting, rabbit polyclonal anti-CPY
antiserum (1:10,000) and horseradish
peroxidase-conjugated anti-rabbit IgG
secondary antibodies (1:100,000) wereused.
(A) (B) Lane 1, molecular marker(phosphorylase b, 97.4 kDa; serum
albumin, 66.2 kDa; ovalbumin, 45.0
kDa; carbonic anhydrase, 31.0 kDa);
lane 2, the solution after activation (31µg); lane 3, the eluate fromTOYOPEARL Butyl-650M column (3.2µg); lane 4, the supernatant after heat
treatment (2.6 µg).

M. Makino, et al.

35

Table 2 Summary of purification of recombinant wild-type CPY
Each parameter was calculated based on the anilidase activity toward BTpNA.

in the folding; however, cysteine at thisrelease of L-leucine at room temperature.

position could be an exception.

The anilidase activity was assayed at room
temperature using BTpNA as a substrate byHydrolase activity of soluble mutants

measuring the rate of release of p-nitroaniline.

The peptidase and anilidase activities ofCompared with wild-type CPY, both mutants

the soluble proteins, Q228C and K314C, were

exhibited lower hydrolase activities toward Z-

measured.

The activity toward Z-Phe-Leu-

Phe-Leu-OH and BTpNA (Table 3(A)). This

OH was determined by measuring the rate ofresult suggests that the replacements of
Gln228 and Lys314 would affect theirhydrolase activities, while the protein folding
was not influenced by the replacement.
Table 3(B) shows the summary ofhydrolase activities of wild-type CPY, P315S,
and P315G.

The relative ratios of thespecific peptidase activities of P315S and
P315G to that of WT were 51 and 55,
respectively.

This result indicates that

P315S and P315G lose their peptidase
activities toward Z-Phe-Leu-OH by half thanthat of WT. On the other hand, the relative
Fig. 5 Western blotting of mutant
CPYs(A)
Cys-introduced
mutants
expressed in yeast cells (1) andactivated and purified (2). (B)Tyr225- and Pro315-substituted
mutants expressed in yeast cells (1)and activated and purified (2).

ratio of specific anilidase activity of P315S
and P315G to WT were 61 and 170,
respectively.

This result shows that the

specific activity of P315G was slightly higher
than that of WT, while that of P315S

36

V-shape helix of yeast carboxypeptidase Y

decreased to approximately 60% of that of WT.

performed.

Table 4 shows the kinetic

Therefore, the replacement of Pro315 may

parameters for P315G-catalyzed hydrolyses ofinfluence catalysis and P315G may alter its

each substrate.

substrate preference.

Phe-Leu-OH and Z-Phe-Pro-OH, were used

Synthetic Z-dipeptides, Z-

for evaluating the hydrolysis activity ofKinetic analysis of P315G

peptide substrates.

The kcat/Km values ofIn order to investigate catalytic activity of

P315G for both substrates were significantly

P315G in derails, kinetic analysis was

reduced as compared with wild-type CPY.

Table 3 Relative specific activity of WT and soluble mutant CPYs toward ZPhe-Leu-OH and BTpNA(A) Cys-introduced mutants (Q228C, K314C). (B) Pro315-subsituted
mutants (P315S and P315G). The activities of recombinant WT for each
substrate were set as 100% active. aEnzyme reaction was performed with
1 mM Z-Phe-Leu-OH at pH 7.0 at room temperature and the amount ofbreleased L-Leu was detected by U-HPLC.
Enzyme reaction was
performed with 0.3 mM BTpNA at pH 7.0 at room temperature and A410 wasmonitored spectrophotometrically.

M. Makino, et al.

37

The kcat values of P315G for Z-Phe-Leu-OH

efficiency of P315G was much lower than thatand Z-Phe-Pro-OH were 3.2 and 0.23,

of WT, due to its remarkable reduction of therespectively, while those of WT were 21 andkcat value as compared with WT.

1.0, respectively.

Thus, the decrease inother hand, the kinetic parameters of P315G

kcat/Km of P315G for those substrates wasfor BTpNA were similar to those of wild-typeascribed to the decrease in kcat.

CPY. These results show that the anilidase

On theIn the hydrolysis of Ac-Tyr-OEt, the Km

activity is maintained in P315G, although thevalue of P315G was slightly decreased

catalysis of P315G in the hydrolysis ofrelative to WT.

peptides and esters is significantly reduced in

Nevertheless, the catalytic

Table 4 Kinetic parameters of recombinant wild-type CPY and
P315G in the hydrolysis of the dipeptide substrates (A), anilide
substrate (B), and ester substrate (C) at room temperature
Each parameter is expressed as mean ± SD calculated from thedata of multiple experiments.

38

V-shape helix of yeast carboxypeptidase Y

facts that V-shape helix lies over the active site

comparison with wild-type enzyme.

cavity and that S3 and S5 subsites consist ofpart of the residues at V-shape helix, our data
could demonstrate the involvement of V-shape

Discussion

helix in catalysis.
According to the refolding experiments,

In generally, proline and glycine residues

the propeptide region of CPY acts as anact as secondary structure breakers: the side

intramolecular chaperon and denatured pro-chain of proline restrains its backbone and

CPY is refolded with correct disulfide bondproline has the lowest conformational entropy,

formation (30,31).

and glycine has a high flexibility because ofHence, CPY has a highability to renature spontaneously.

However,

its hydrogen substituent (32-35).

In thisin this study, it was found that the presence ofstudy, the various phenotypes of each mutant

additional only one cysteine at or near the tip

which Pro315 was replaced by other aminoof V-shape helix caused a severe misfoldingacid reveal that the fluctuation of the(Fig. 5(A)). Furthermore, our data presented

backbone of residue 315 caused by thein Fig. 5(B) that replacement of Tyr225

substitution of Pro315 have apparent effects

positioned at the tip of V-shape helix by otheron the protein folding (Fig. 5(B)), hydrolase

amino acids led to aggregation of CPY

activitymolecule show that the residue at the tip of therecognition (Table 4).

V-shape helix contribute to the formation ofwould influence the construction of V-shapeproper conformation of CPY.

helix because the residue 315 is proximate toThus, our

(Table3(B)),

andsubstrate

This fluctuation

results demonstrate that V-shape helix would

V-shape helix.

be involved in the folding of CPY molecule.

functional CPY to maintain properly the

To date, there has been little recognition ofconfiguration of the amino acid residues

the residues that are involved in catalysis
besides these residues at the substrate bindingsites in CPY.

On the other hand,Thus, it is necessary foraround V-shape helix.
In conclusion, we propose that V-shape
helix plays a role in constructing a functional

experimental results of this study showed thatCPY.

the residues, Gln228, Lys314, and Pro315,

insertion domain, part of structure elements

which are positioned at or near V-shape helix

that deviate from the core domain, is a key

and are distant away from the active site, areregionfold

serineinvolved in the hydrolysis activity of CPY

carboxypeptidase as well as lipases.

This(Table 3(A)(B)).

study will provide a new insight into the

In combination with the

And it was demonstrated that the

in

α/β

hydrolaseM. Makino, et al.

39

folding process and diversity of substratecarboxypeptidase. Biochemistry 33,

recognition mechanism in α/β hydrolase fold

11106-11120
6.

proteins.

Liao, D. I., Breddam, K., Sweet, R.
M., Bullock, T., and Remington, S. J.
(1992) Refined atomic model ofwheat serine carboxypeptidase II at

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Communicated by Takahashi Saori