Amphipathic property of free thiol group contributes to an increasein the catalytic efficiency of carboxypeptidase Y Joji Mima, Giman Jung, Takuo Onizuka, Hiroshi Ueno and Rikimaru Hayas
Trang 1Amphipathic 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 Km whereas hydrophobic amino acids in this
position tended to decrease Km 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 kcatvalues 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 kcatvalues 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] Athird
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 Anumber 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: mima@kais.kyoto-u.ac.jp
Enzyme: carboxypeptidase Y (EC 3.4.16.5).
(Received 15 January 2002, revised 13 May 2002,
accepted 15 May 2002)
Trang 2designed 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
M A T E R I A L S A N D M E T H O D S
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 TransformerTM
site-directed mutagenesis kit was purchased from Clontech, Palo
Alto, CA, USA The Taq dyedeoxyTM 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) leu 2-3, 112 ura 3-52 hi 4-519) 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¢); a 26-mer as the selection primer (5¢-ACTACAAAATGAGCTCCCTCGC GCGT-3¢) to introduce a PvuII restriction site into plasmid pTSY3 for selection; and a 20-mer sequence primer for
AG-3¢) The mutation was performed with a Transformer site-directed mutagenesis kit as described by Deng & Nickoloff [14] The DNAsequence reaction was performed with a Taq dyedeoxy terminator cycle sequencing kit Yeast strain SEY2202 was transformed by the lithium acetate method [15] E coli JM109 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 4C, were loaded on to a hydroxylapatite column equilibrated with 75 mM sodium phosphate buffer, pH 6.8, and both wild-type carboxypeptidase Y and mutant carboxypepti-dase Y were eluted with 150 mM 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 lMin 10 mMsodium phosphate,
pH 7.0
Kinetic characterization Peptidase activities were measured in 50 mM Bistris buffer/1 mM EDTA , pH 6.5, at 25C Enzyme concen-trations of the wild-type and mutant carboxypeptidase Y
in the hydrolysis reaction were 32.8 nM Substrate con-centrations were 0.02–20 mM The reaction was termin-ated and deproteinized by adding 330 lL 0.4M
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-His diad The sulfur atom of Cys341 is located within 5 A˚ of the
imidazole ring of His397.
Trang 3Calculation 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 (k2 k3); therefore, Km Ks and
k2 kcatare assumed [20] The energy level, DG, of each
enzyme state was calculated from the following
thermody-namic equations [20] and is shown relative to the free
enzyme defined as DG¼ 0 (Table 3):
DGs¼ RT lnKs
RT lnKm;
DG‡¼ RT lnðkBT=hÞ RT lnk2
RT lnðkBT=hÞ RT lnkcat;
DG‡T¼ RT lnðkBT=hÞ RT lnðk2=KsÞ
RT lnðkBT=hÞ RT lnðkcat=KmÞ;
DDG‡¼ DG‡ðmutantÞ DG‡ðwild-typeÞ
where DGs is the binding energy of the substrate to the
enzyme, DGis the activation energy in the chemical steps of
bond making and breaking, DG‡Tis 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, kBis the Boltzmann’s constant, h is Planck’s constant
R E S U L T S
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-X1-Leu-OH and Cbz-X2 -Phe-OH, where X1was a Gly or Phe residue and X2was a Gly or Ala residue (Table 2) Mutant enzymes had reduced
kcat/Kmvalues 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 kcat/Km values with Cbz-Gly-Leu and Cbz-Gly-Phe, although C341G exhibited higher kcat/Kmvalues than the other mutants with Cbz-Gly-Leu The kcat values of C341G, C341S and C341F were largely higher than that of the wild-type Anearly identical profile was observed for Kmvalues 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)
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.
Trang 4Cbz-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 Kmvalues 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 kcat values (20–90-fold decreased compared
with the kcatvalue 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 kcat/Kmvalues 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]
D I S C U S S I O N
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 kcat/Km 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 kcatvalues 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
kcat 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 Km 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 Km values (Table 2) It was also assumed in a previous report [7] that the higher Kmvalues 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)
Ahydrophilic 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 Km Mutant enzymes in which Cys341 was replaced with Glu and Gln had higher Km 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)1Æm M )1 ) Wild-type Cbz-Gly-Leu 3.2 0.83 3.8
Trang 5carboxypeptidase Y have similar Km 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 kcat 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 kcat This suggests
an additional role for this thiol group
Free-energy parameters derived from the kinetic analysis
are summarized in Table 3 for the wild-type and C341V
mutant enzymes This result can be visualized in the form of
a diagrammatic scheme shown in Fig 3 Acharacteristic of
C341V is its increased activation energy, DG, 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
DGcompared 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
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)
Such an alteration in the side chain of Cys341 in the 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 would be used to create a
substrate-Table 3 Gibbs free energies (kJÆmol)1) of complexes of wild-type
carboxypeptidase Y and C341V mutant DG S is algebraically negative
and DGand DG‡T positive The activation energy (DG) of wild-type
carboxypeptidase Y is lowered by DDG compared with that of
C341V.
Substrate Enzyme DG ‡
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.
Trang 6binding 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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