Native subtilisin Karlsberg and modified subtilisin 72 as effective catalysts of peptide bond formation in organic media

Article abstract of DOI:10.1134/S1068162006020026

The activity and stability of native subtilisin Karlsberg and subtilisin 72 and their complexes with sodium dodecyl sulfate (SDS) in organic solvents were studied. The kinetic constants of the hydrolysis of specific chromogenic peptide substrates Z-Ala-Ala-Leu-pNA and Glp-Ala-Ala-Leu-pNA by the subtilisins were determined. It was found that the subtilisin Karlsberg complex with SDS in anhydrous organic solvents is an effective catalyst of peptide synthesis with multifunctional amino acids in positions P¹ and P′₁ (Glu, Arg, and Asp) containing unprotected side ionogenic groups. Pleiades Publishing, Inc., 2006.

Full text of DOI:10.1134/S1068162006020026

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2006, Vol. 32, No. 2, pp. 116–121. © Pleiades Publishing, Inc., 2006.
Original Russian Text © O.M. Anikina, T.A. Semashko, E.S. Oksenoit, E.N. Lysogorskaya, I.Yu. Filippova, 2006, published in Bioorganicheskaya Khimiya, 2006, Vol. 32, No. 2,
pp. 130–136.
Native Subtilisin Karlsberg and Modified Subtilisin 72
As Effective Catalysts of Peptide Bond Formation
in Organic Media
O. M. Anikinaa, T. A. Semashkob, E. S. Oksenoitb,
E. N. Lysogorskayab, and I. Yu. Filippovab, 1
Faculty of Bioengineering and Bioinformatics, Moscow State University, Vorob’evy gory, Moscow, 119992 Russia
a
b
Faculty of Chemistry, Moscow State University, Vorob’evy gory, Moscow, 119992 Russia
Received July 6, 2005; in final form, July 13, 2005
Abstract—The activity and stability of native subtilisin Karlsberg and subtilisin 72 and their complexes with
sodium dodecyl sulfate (SDS) in organic solvents were studied. The kinetic constants of the hydrolysis of spe-
cific chromogenic peptide substrates Z-Ala-Ala-Leu-pNA and Glp-Ala-Ala-Leu-pNA by the subtilisins were
determined. It was found that the subtilisin Karlsberg complex with SDS in anhydrous organic solvents is an
'
effective catalyst of peptide synthesis with multifunctional amino acids in positions P1 and P1 (Glu, Arg, and
Asp) containing unprotected side ionogenic groups.
Key words: enzymatic peptide synthesis, organic solvents, SDS–subtilisin Karlsberg comple
DOI: 10.1134/S1068162006020026
INTRODUCTION
RESULTS AND DISCUSSION
Subtilisin Karlsberg is a widely distributed and
commercially available enzyme with a wide substrate
specificity; it is very close, but not identical to
subtilisin 72 [2].
The use of proteases as catalysts of the peptide bond
formation in polar organic media is of fundamental
importance for the design of new methods of enzymatic
synthesis.2 The application of known enzymes to the
catalysis of the peptide bond formation is often limited
by their substrate specificity. Therefore, a search for
new biocatalysts suitable for the purposeful enzymatic
peptide synthesis is topical. It has earlier been shown in
our laboratory that subtilisin 72 (both native and modi-
fied) is not only highly hydrolytically active, but can
also catalyze the formation of peptide bond in organic
media between various amino acids, including those
that do not correspond the enzyme specificity in hydro-
lytic reactions [1]. In this work, we set a task to reveal,
whether a high activity and wide spectrum of substrate
specificities of subtilisin 72 are its unique properties or
they are also inherent of subtilisin Karlsberg.
We chose two subtilisin-specific peptide substrates
Z-Ala-Ala-Leu-pNA (I) [3] and Glp-Ala-Ala-Leu-pNA
(II) [4] for a comparative analysis of the hydrolytic
capacity of subtilisin 72 and subtilisin Karlsberg prepa-
rations. These substrates are close structural analogues,
and they only differ in N-terminal residues, position P4
according to the Schechter and Berger nomenclature [5].
The replacement of the bulky aromatic benzyloxycarbo-
nyl group in (I) by a hydrophilic residue of pyroglutamic
acid in (II) affected the substrate solubility: water–
organic mixtures containing at least 20% DMSO or
DMF should be used to dissolve (I), whereas (II) is much
more soluble in water, which allowed us to decrease the
concentration of organic solvents in system to 1–2%.
Thus, a comparative use of substrates (I) and (II) permits
us, first, to consider the effect of structurally various sub-
stituents in position P4, which is crucial for the specific-
ity of the whole subtilisin family, on the subtilisin activ-
ity and, second, to study the effect of organic solvents on
the enzymatic activity.
1
Corresponding author; phone: +7 (495) 939-5529; e-mail: irfil-
ipp@genebee.msu.su.
2
Abbreviations: Ded, N-[2-(2,4-dinitropenylamino)ethyl]amide;
Glp, pyroglytamyl; pNA, para-nitroanilide; SDS, sodium dode-
cyl sulfate; and Z, benzyloxycarbonyl. All amino acids belong to
the L series.
116

NATIVE SUBTILISIN KARLSBERG AND MODIFIED SUBTILISIN 72
117
Table 1. Kinetic constants of the subtilisin Karlsberg-induced hydrolysis of Z-Ala-Ala-Leu-pNA (I) and Glp-Ala-Ala-Leu-pNA
(II)*
Content
of organic
solvent
kcat/Km,
Km, mM
kcat, s–1
s–1 mM–1
Enzyme
Substrate
DMF
DMSO
DMF
DMSO
DMF DMSO
(vol %)
Subtilisin
Karlsberg
Z-Ala-Ala-Leu-pNA (I)
Glp-Ala-Ala-Leu-pNA (II)
20
2
0.19 0.02 0.14 0.01 70.9 1.7 100
1
2
373.2 714.3
357.2 615.4
0.13 0.01 64.3 1.4
0.25 0.02 104 19
80
77
0.18 0.02
4.0 1.0
Subtilisin 72 Z-Ala-Ala-Leu-pNA (I)
Glp-Ala-Ala-Leu-pNA (II)
20
2
9
25.7 308.4
–
69
8
–
277.2
–
0.25 0.02
* A dash designates the lack of experimental data.
One can see from Table 13 that the Km values for must be highly stable in both aqueous media and in
both substrates in the case of subtilisin Karlsberg are anhydrous organic solvents.
practically the same in the media containing 2 and 20%
The subtilisin Karlsberg–SDS complex was
of organic solvents (DMF or DMSO). On the other
obtained under the conditions earlier described for sub-
hand, substantial differences were observed in Km val-
ues for the hydrolysis of substrates (I) and (II) by sub-
tilisins BPN’ and 72 [8, 9].
The activity of the subtilisin Karlsberg–SDS com-
plex was monitored by the cleavage of the same chro-
mogenic substrates (I) and (II) after the dilution of an
aliquot of the complex in ethanol with a large volume
of water. The values of specific activity of the resulting
complex in DMF and DMSO are shown in Table 2.
tilisin 72. The Km value is approximately 4 mM for the
hydrolysis of (I) in 20% DMF, whereas the Km value is
0.25 mM for (II) when the DMF content decreased to
2%. In DMSO, this value decreased for substrate (I),
but it still remains twice as high in 20% DMSO in com-
parison with subtilisin Karlsberg. This may be associ-
ated with a lower solvent–substrate competition for the
enzyme binding site. In the case of subtilisin Karlsberg,
the kcat value is practically equal for both substrates and
does not depend on the concentration of organic solvent
in system. The pass from DMF to DMSO causes only
insignificant increase in kcat.
An analysis of the results suggests that, according to
the hydrolysis of (I) in 20% DMF, the activities of the
native subtilisin Karlsberg and its SDS complex are
equal. No stabilizing effect of complex formation was
observed in DMSO, as the activity of the native subtil-
isin Karlsberg was two times higher than the activity of
the complex. According to the hydrolysis of (II), the
specific activity of subtilisin Karlsberg in the complex
was rather high, although it was somewhat lower than
that of the native enzyme.
It seems interesting to compare proteolytic coeffi-
cients kcat/Km of subtilisins Karlsberg and 72 in DMF
and DMSO. Obviously, in the case of subtilisin Karls-
berg, their values insignificantly differ the two sub-
strates both in DMF and DMSO, but, unlike DMF, the
kcat/Km ratio in DMSO is twice as high. This fact sup-
ports a lower denaturing effect of DMSO for the
enzyme. Unlike subtilisin Karlsberg, the kcat/Km ratio
for subtilisin 72 differs in DMF by one order of magni-
tude for the two substrates.
We studied changes in the specific activity of the
subtilisin Karlsberg–SDS complex during its storage
with the goal to determine the effect of complex forma-
tion on the enzymatic stability. The activity of the
native enzyme in 0.05 M Tris-HCl buffer, pH 8.2, con-
taining 2 mM CaCl2 (buffer A) in the reaction of (II)
hydrolysis at the initial time point was taken as 100%.
The subtilisin Karlsberg–SDS complex retained 75%
of its activity when stored in ethanol at +4°ë for
A comparison of kinetic parameters of the substrate
hydrolysis with subtilisin Karlsberg and subtilisin 72
indicates noticeable differences in enzymatic properties
of these closely relative enzymes.
Table 2. Specific activity of the SDS-subtilisin Karlsberg
The preparation of noncovalent complexes of
enzymes with detergents and polyelectrolytes is one of
the most effective methods for the enzyme adaptation
to functioning in polar organic solvents [6, 7]. We used
this approach for the preparation of subtilisin Karls-
berg–SDS complex. According to [8], such complexes
complex in the presence of DMF and DMSO
Content
of organic
solvent
Specific activity*,
µmol/OU280 min
Substrate
DMF
DMSO
(vol %)
Z-Ala-Ala-Leu-pNA
20
2
14 (14) 33 (68)
52 (75) 57 (100)
3
The results of active site titration of subtilisin Karlsberg and 72
were taken into account in the calculation of kinetic constants
(see the Experimental section); the concentrations of active sites
of subtilisins Karlsberg and 72 were 50 and 75%, respectively.
Glp-Ala-Ala-Leu-pNA
* The activity of native enzyme is given in parentheses.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 2 2006

118
ANIKINA et al.
Table 3. The peptides synthesized using the SDS-subtilisin Karlsberg complex*
Acylating component
Amino component
H-Phe-pNA
Product
Time, h
Yield, %
Z-Ala-Ala-Leu-OCH3
Z-Ala-Ala-Leu-OCH3
Z-Ala-Ala-Leu-OCH3
Z-Thr-Ala-Thr-OCH3
Z-Ala-Ala-Ile-OCH3
Z-Ala-Ala-Arg-OCH3
Z-Ala-Ala-Glu-OH
Z-Ala-Ala-Glu-OH
Z-Ala-Glu-OH
Z-Ala-Ala-Leu-Phe-pNA (1)
Z-Ala-Ala-Leu-Arg-pNA (2)
Z-Ala-Ala-Leu-Asp-pNA (3)
Z-Thr-Ala-Thr-Asp-pNA (4)
Z-Ala-Ala-Ile-Phe-pNA (5)
Z-Ala-Ala-Arg-Arg-pNA (6)
Z-Ala-Ala-Glu-Arg-pNA (7)
Z-Ala-Ala-Glu-Phe-Phe-Ded (8)
Z-Ala-Glu-Phe-Phe-Ded (9)
Z-Ala-Glu-Phe-Ded (10)
1
1
95
H-Arg-pNA
H-Asp-pNA
H-Asp-pNA
H-Phe-pNA
H-Arg-pNA
H-Arg-pNA
H-Phe-Phe-Ded
H-Phe-Phe-Ded
H-Phe-Ded
90
24
1
60
98 (90)**
24
1
0
70
85
30
0
1
24
24
24
24
Z-Ala-Glu-OH
0
Z-Ala-Glu-OH
H-Phe-pNA
Z-Ala-Glu-Phe-pNA (11)
60
Notes: * Coupling conditions: 30 mM acylating and amino components, 2 µM SDS-subtilisin in ethanol, 20°C.
** The preparative yield of peptide is given in parentheses.
2 months, whereas the native subtilisin Karlsberg was
completely inactivated in bufferA after one-day storage
at room temperature. The stabilities of the native subtil-
isin 72 and its SDS complex are also shown in Fig. 1 for
comparison. One can see that the stability of the SDS–
subtilisin 72 complex was also higher than that of the
native enzyme.
The subtilisin Karlsberg–SDS complex was tested
in the reactions of enzymatic peptide synthesis in the
media with a high concentration of organic solvents.
As a model reaction, we chose the reaction of
Z-Ala-Ala-Leu-Phe-pNA formation from Z-Ala-Ala-
Leu-OMe and Phe-pNA, which was thoroughly studied
for the subtilisin 72–SDS complex [9]. It proceeded
according to the scheme:
Z–Ala–Ala–Leu–OH
Phe–pNA
Z–Ala–Ala–Leu–OMe + E
[Z–Ala–Ala–Leu–O–E]
Z–Ala–Ala–Leu–Phe–pNA
MeOH
EtOH
Z–Ala–Ala–Leu–OEt
The structure and length of the starting compounds
corresponded to the subtilisin specificity, which
ensured their good binding to the enzyme active site.
Equimolar amounts of amino and carboxy components
were introduced into the reaction. The [E]/[S] molar
ratio was 1 : 5 × 103. The reaction proceeded in anhy-
drous polar organic solvents: DMF or DMSO and etha-
nol. The dependence of the product yield on the
enzyme concentration was studied in the model reac-
tion of Z-Ala-Ala-Leu-Phe-pNA (1) formation in the
We also carried out a similar model reaction in the
medium containing DMSO with the goal to determine
the effect of the nature of organic solvents on the effi-
ciency of the peptide bond formation catalyzed by the
subtilisin Karlsberg–SDS complex. The yield of Z-Ala-
Ala-Leu-Phe-pNA at the complex concentration of
1 µM achieved 90% in DMSO, whereas it was only
50% in DMF.
The study of the synthase activity of the subtilisin
Karlsberg–SDS complex by the example of the model
presence of the subtilisin Karlsberg–SDS complex in reaction showed that it is a rather effective catalyst of
the peptide bond formation. With Z–Ala–Ala–Leu–
OCH3 as an acylating component and the SDS–subtili-
sin complex as a catalyst, we synthesized tetrapeptides
containing residues of unprotected ionogenic multi-
DMF (Fig. 2, curve 1). The maximal yield (98%) was
achieved after 0.5 h at the enzyme concentration
exceeding 5 µM. For a comparison, the data on the
efficiency of the use of subtilisin 72–SDS complex in a
similar reaction are shown in this figure (Fig. 2,
curve 2) [9]. The comparison demonstrates that the
synthase activity of the subtilisin Karlsberg–SDS com-
plex was slightly higher in this reaction than that of a
similar complex of subtilisin 72.
'
functional acids, Arg and Asp, in position P1 (Table 3).
A high efficiency of the complex was observed in the
synthesis of peptide (2), whose content in the reaction
mixture was 90% after 1 h, whereas only 60% of pep-
tide (3) was obtained after 24 h. At the same time, the
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 2 2006

NATIVE SUBTILISIN KARLSBERG AND MODIFIED SUBTILISIN 72
Residual specific activity, %
119
synthesis of peptide (4) with Asp-pNA as an amino
component proceeded quantitatively already after 1 h.
Apparently, the replacement of Leu by a Thr residue in
position P1 affected the efficiency of the synthesis. On
the other hand, the replacement of Leu by a β-branched
Ile residue in position P1 did not result in the formation
of Z-Ala-Ala-Ile-Phe-pNA (5) even after 2 days. This
agrees well with the results on the subtilisin Karls-
berg specificity in the hydrolysis of the substrates, in
which amino acid residues preferable in position P1
can be arranged in the following order: Leu > Phe >
Val >Ile [10].
100
80
60
40
20
(a)
1
2
By the example of the peptide (6) synthesis, we
showed an ability of effective use of the subtilisin
Karlsberg–SDS complex as the catalyst for the peptide
bond formation between two Arg residues. The yield of
the target product achieved 70% after 1 h, whereas it
did not exceed 30% after 17-h reaction catalyzed by the
subtilisin 72–SDS complex [11].
40 60 80 100 120 140
0
20
(b)
100
80
60
40
20
1
2
We synthesized tetrapeptide (7) containing oppo-
sitely charged amino acid residues Glu and Arg in posi-
'
tions P1 and P1 in 85% yield for 1 h. The fundamental
possibility of this synthesis can be explained by a spec-
ificity of the biocatalytic system: anhydrous organic
solvents allow the avoidance of the charge appearance
on ionogenic groups, whereas electroneutral hydropho-
bic amino acid residues are good bound by the subtili-
sin active site.
0
100
500
Time, h
Fig. 1. The time dependence of activities of (a) native (1)
subtilisin Karlsberg and (2) subtilisin 72 in Tris-HCl buffer
(pH 8.2) and (b) of their SDS complexes at +4°ë.
The synthesis of peptide (8) was much less success-
ful: the peptide yield was only 30% after 24 h. This may
be explained by the use of strongly hydrophobic and
sterically voluminous dipeptide Phe-Phe-Ded as an
amino component. The use of the same amino compo-
nent and a truncated dipeptide analogue of Z-Ala-Ala-
Glu-OH did not result in product (9) even after 3 days.
Probably, the presence of a 2,4-dinitrophenylethylene-
diamine residue in the substrate molecule dramatically
affect the substrate–enzyme binding. The syntheses of
peptides (10) and (11) support this hypothesis: the yield
of Z-Ala-Glu-Phe-pNA was 60% for a day, whereas no
formation of Z-Ala-Glu-Phe-Ded was observed.
We carried out a preparative synthesis of Z-Thr-Ala-
Thr-Asp-pNA (4), a chromogenic highly specific sub-
strate for the caspaselike protease of the plant origin
[12], for the demonstration of catalytic capacity of the
tested modified enzyme in peptide synthesis.
The peptide was synthesized by the kinetically con-
trolled reaction from two fragments using the subtilisin
Karlsberg–SDS complex in ethanol.
cipitation with ether from DMF solution. When the
whole reaction mixture was dissolved in DMF, the sub-
tilisin Karlsberg–SDS complex was precipitated and,
therefore, was easily separated by centrifugation. The
Yield, %
100
80
60
40
20
1
2
Z-Thr-Ala-Thr-OCH3 + H-Asp-pNA
Z-Thr-Ala-Thr-Asp-pNA.
0
1
2
3
4
5
6
7
8
E, µM
The starting compounds were dissolved in DMSO
up to the concentration of 30 mM; the [E] : [S] was
1 : 5000. The enzyme was used as the complex with
SDS dissolved in ethanol. The analytical peptide yield
achieved 98% for 1 h. The peptide was purified by pre-
Fig. 2. The yields of Z-Ala-Ala-Leu-Phe-pNA in the syn-
theses catalyzed by SDS complexes of (1) subtilisin Karls-
berg and (2) subtilisin 72 in ethanol. The synthetic condi-
tions: [S] was 30 mM, 3 : 7 DMF–ethanol (vol), 0.5 h.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 2 2006

120
ANIKINA et al.
yield of the target peptide was 90%. The product was were registered (a nonenzymatic substrate hydrolysis).
characterized by HPLC and amino acid analysis.
A subtilisin solution in 0.1 M acetate buffer (pH 5.05,
100 µl, 7.6 mg/ml of protein) was added to the resulting
solution, and the fall in optical absorption was registered.
The active site concentration in the subtilisin preparation
was calculated using the N-trans-cinnamoylimidazole
molar absorption ε310 = 23 800 M–1 cm–1.
The preparation of the subtilisin Karlsberg–SDS
complex. A 7 mM solution of sodium dodecyl sulfate
in water (0.5 ml) was added to a solution of the subtili-
sin Karlsberg preparation (2 mg, 70 nmol of protein) in
1 mM CaCl2 (1 ml, pH 5.5). The reaction mixture was
stirred for 5 min, and kept for 30 min at room tempera-
ture. The resulting precipitate was separated by centrif-
ugation for 15 min at 12 000 rpm, dissolved in ethanol
(1 ml), and the optical absorption of supernatant was
measured at 280 nm.
The determination of subtilisin enzymatic activ-
ity by hydrolysis of Glp-Ala-Ala-Leu-pNA in DMF
and DMSO. A 20 mM substrate solution in anhydrous
DMF (or DMSO) (10 mg/ml, 50 µl) was added to a
0.05 M Tris-HCl buffer (pH 8.2, 2.5 ml), and the solu-
tion was incubated for 10 min at 37°ë. A solution of the
tested enzyme (0.1 mg/ml, 10 µl) was added, and the
mixture was incubated at 37°ë until the absorption
value achieved 0.1–0.4 at 410 nm. The reaction was
quenched by addition of 50% acetic acid (0.5 ml) and
A410 was measured. The order of the enzyme and acetic
We demonstrated a fundamental possibility of using
the resulting substrate for testing the activity of the
caspaselike protease from tobacco leaves in prelimi-
nary experiments.
To summarize, according to the analysis of results,
the subtilisin Karlsberg–SDS complex is an effective
catalyst of peptide formation in a mixture of anhydrous
organic solvents.
EXPERIMENTAL
Serine proteases from Bacillus licheniformis, subtil-
isin Karlsberg (EC 3.4.21.62, M 23 000 Da), and CaCl2
were purchased from Sigma (United States). Subtilisin
72 was isolated from a commercial preparation of the
cultural fluid of Bacillus subtilis, strain 72
(M 27500 Da) and purified as described in [2]. Aceto-
nitrile for HPLC of special purity grade containing no
more than 0.01% water was from Lekbiopharm (Rus-
sia); DMF of the analytical purity grade was addition-
ally purified as described in [2]. Acetic acid was from
Reakhim (Russia), and triethylamine of the analytical
purity grade (Reakhim, Russia) was additionally dis-
tilled as described in [13]. Trifluoroacetic acid of the
analytical purity grade was from Fluka Chemie (Swe-
den).
Amino acid and peptide derivatives were synthe- acid addition was reverse in reference samples. The
sized in our laboratory by standard procedures [14]. specific activity was calculated by the following for-
The Z-Ala-Ala-Leu-pNA (I) and Glp-Ala-Ala-Leu- mula:
pNA (II) substrates were synthesized by the procedures
(A410 – A4c10 )Vsample
[3] and [4], respectively.
-----------------------------------------------
,
Spectrophotometric measurements were carried out
on a Specord UV VIS (Germany), a DW-2000TM a UV
VIS SLMAminco (United States), or a Pharmacia LKB
Ultrospec III (Sweden) spectrophotometer.
Amino acid analysis was obtained on an automatic
amino acid Hitachi 835 analyzer (Japan) after acidic
hydrolysis with 5.7 M HCl at 105°ë in vacuumated
ampoules for 24 and 48 h.
Peptides were analyzed on an Altex Model 110 A
liquid chromatograph (United States). The elution with
0.1% ëF3COOH in a linear gradient of acetonitrile
(from 10 to 70% for 26 min, A) at a flow rate of
1 ml/min on a Microsorb-MV C8 column (4.6 × 250 mm,
Rainin Instrument Company, Inc.) and in a linear gradi-
ent of acetonitrile (from 10 to 50% for 26 min, B) on a
Nucleosil C18 (4.6 × 250 mm, Biochimmak, Moscow)
column. The detection was carried out at 220 and
280 nm. The compositions of reaction mixtures were
calculated without corrections to the differences in
molar absorption coefficients of components.
A280tVE × 8.9
where A4c10 is the absorption of reference solution at
410 nm; A410, the absorption of the tested solution at
410 nm; A280, the absorption of enzyme solution at
280 nm; Vsample, the sample volume (ml); t, the reaction
time (min); VE, the volume of the enzyme sample (ml);
and 8.9, the millimolar absorption coefficient of
p-nitroaniline (mM–1 cm–1).
The enzyme amount necessary for the cleavage
from substrate of 1 µmol of p-nitroaniline for 1 min
under the described conditions is taken as 1 activity
unit.
The determination of subtilisin enzymatic activ-
ity by the Z-Ala-Ala-Leu-pNA hydrolysis in DMF
and DMSO. A 0.05 M Tris-HCl buffer (pH 8.2, 2.0 ml)
was added to a thermostatted at 37°ë solution of Z-Ala-
Ala-Leu-pNA in DMF or DMSO (0.5 mg/ml, 0.5 ml).
A solution of the enzyme tested (0.5 mg/ml of protein,
Titration of the subtilisin Karlsberg active sites. 10 µl) was then added and the mixture was incubated at
A solution of N-trans-cinnamoylimidazole in acetoni- 37°ë until the development of yellow color. The reac-
trile (2 mg/ml, 25 µl) was added into a quartz cuvette tion was quenched by addition of 50% acetic acid
containing 0.1 M acetate buffer (3 ml), pH 5.05. (0.5 ml), and A410 was measured. The order of the
Changes in optical absorption of the solution at 310 nm enzyme and acetic acid addition was reverse in refer-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 2 2006

NATIVE SUBTILISIN KARLSBERG AND MODIFIED SUBTILISIN 72
121
ence samples. The specific activity was calculated by 29.1 min. The amino acid analysis (nmol): Ala 8.2,
the above formula.
Glu 4.0, and Arg 3.9.
The activity of the subtilisin Karlsberg–SDS com-
plex dissolved in ethanol was similarly measured by the
addition of the solution of subtilisin Karlsberg–SDS
complex in ethanol (0.66 mg/ml of protein) to the reac-
tion mixture.
Z-Ala-Ala-Leu-Arg-pNA (2), Z-Ala-Ala-Leu-
Asp-pNA (3), and Z-Ala-Ala-Glu-Phe-Phe-Ded (8)
were synthesized similarly. The HPLC RTs of the pep-
tides (2), (3), and (8) in gradient B were 21.9, 24, and
27.7 min, respectively. The amino acid analysis (nmol)
for peptide (2): Ala 4.1, Leu 1.9, and Arg 2.0; for pep-
tide (3): Ala 6.4, Leu 3.0, and Asp 3.3; and for peptide
(8): Ala 6.1, Glu 3.1, and Phe 6.2.
Kinetic studies of the subtilisin Karlsberg-
induced hydrolysis of Glp-Ala-Ala-Leu-pNA. Solu-
tions of 0.05 M Tris-HCl buffer (pH 8.2) containing
2 mM CaCl2 (2.5 ml) and Glp-Ala-Ala-Leu-pNA in
DMF or DMSO (50 µl) were placed into a thermostat-
ted cell (37°ë) to a final substrate concentration of 0.1–
0.75 mM, and the enzyme solution in Tris-HCl buffer
(pH 8.2, 50 µl) was added. The initial reaction rate was
determined spectrophotometrically at 410 nm accord-
ing to the p-nitroaniline formation. The catalytic con-
stant kcat and Michaelis constant Km were determined
by analyzing the reaction rate–substrate concentration
dependence using double reverse Lineweaver–Burk
coordinates.
ACKNOWLEDGMENTS
This work was supported by the Russian Foundation
for Basic Research, project no. 03-03-32847.
REFERENCES
1. Bacheva, A.V., Baibak, O.V., Belyaeva, A.V., Filip-
pova, I.Yu., Lysogorskaya, E.N., Oksenoit, E.S., Lozin-
skii, V.I., and Velichko, T.I., Biokhimiya (Moscow),
2003, vol. 68, pp. 1567–1574.
2. Akparov, V.Kh., Belyanova, L.P., Baratova, L.A., and
Stepanov, V.M., Biokhimiya (Moscow), 1979, vol. 44,
pp. 886–891.
Peptide Synthesis
Z-Ala-Ala-Leu-Phe-pNA (conventional proce-
dure). Ethanol (65 µl) and a solution of the subtilisin
Karlsberg–SDS complex in ethanol (60 µl, 0.66 mg/ml
of protein) were added to a solution of Z-Ala-Ala-Leu-
OMe (5.5 µmol) and Phe-pNA (1.4 mg, 5.5 µmol) in
DMF (50 µl). The reaction mixture was shaken on an
orbital shaker, when periodically taking aliquots (10 µl
each) and analyzing them by HPLC in gradient A. RT
of Z-Ala-Ala-Leu-Phe-pNA (1) was 18.4 min. The
amino acid analysis (nmol): Ala 10.2, Leu 5.6, and
Phe 5.4.
Z-Ala-Glu-Phe-pNA (11) was synthesized in a
similar way. RT of Z-Ala-Glu-Phe-pNA (11) in gradi-
ent B was 26 min. The amino acid analysis (nmol): Ala
3.9, Phe 4.1, and Glu 4.0.
Z-Thr-Ala-Thr-Asp-pNA (4), Z-Ala-Ala-Ile-Phe-
pNA (5), and Z-Ala-Ala-Arg-Arg-pNA (6) were syn-
thesized in a similar manner using DMSO to dissolve
starting reagents. The HPLC RTs of the peptides (4)
and (6) in gradient A were 16 and 24.5 min, respec-
tively. The amino acid analysis (nmol) for peptide (4):
Thr 14.8, Asp 7.0; Ala 6.8; and for peptide (6): Ala 4.6,
Arg 4.4.
3. Lyublinskaya, L.A., Voyushina, T.L., and Stepanov, V.M.,
Bioorg. Khim., 1982, vol. 8, pp. 1620–1624.
4. Lyublinskaya, L.A., Khaidu, I., Balandina, G.N., Filip-
pova, I.Yu., Markaryan, A.I., Lysogorskaya, E.N., Okse-
noit, E.S., and Stepanov, V.M., Bioorg. Khim., 1987,
vol. 13, pp. 748–753.
5. Schechter, I. and Berger, A., Biochem. Biophys. Res.
Commun., 1967, vol. 27, pp. 157–162.
6. Gladilin, A.K. and Levashov, A.V., Biokhimiya (Mos-
cow), 1998, vol. 63, pp. 408–421.
7. Meyer, J., Kendryck, B., and Matsuura, J., Int. J. Pept.
Protein Res., 1996, vol. 47, pp. 177–181.
8. Wangikar, P.P., Michels, P.C., Clark, D.S., and
Dordick, J.S., J. Am. Chem. Soc., 1997, vol. 119,
pp. 70−76.
9. Getun, I.V., Filippova, I.Yu., Lysogorskaya, E.N.,
Bacheva, A.V., and Oksenoit, E.S., Rus. J. Bioorg.
Chem., 1999, vol. 25, pp. 526–531.
10. Perona, J.J. and Craik, C.S., Protein Sci., 1995, vol. 4,
pp. 337–360.
11. Getun, I.V., Filippova, I.Yu., Lysogorskaya, E.N., and
Oksenoit, E.S., J. Mol. Catalysis, B: Enzymatic, 2001,
vol. 627, pp. 1–6.
Z-Ala-Ala-Glu-Arg-pNA (7). A 1 M triethylamine
solution in DMF (7.5 µl) was added to a 400 mM
HCl · Arg-pNA in DMF (18.8 µl), the mixture was 12. Chichkova, N.V., Kim, S.H., Titova, E.S., Kalkum, M.,
Morozov, V.S., Rubtsov, Yu.P., Kalinina, N.O., Talian-
sky, M.E., and Vartapetian, A.B., Plant Cell, 2004,
vol. 16, pp. 157–171.
stirred for 15 min, and the resulting precipitate was sep-
arated by centrifugation. The supernatant was poured
into 400 mM Z-Ala-Ala-Glu-OH solution (18.75 µl),
DMF (30 µl), ethanol (154 µl), and the subtilisin–SDS 13. Gordon, A.J. and Ford, R.A., The Chemist’s Guide, New
York: J. Wiley and Sons, 1974.
complex in ethanol (0.66 mg/ml, 20.5 µl) were added.
The reaction mixture was stirred at 20°ë, and aliquots
(10 µl) were taken out at certain intervals and analyzed
by HPLC in gradient B. The RT of peptide (7) was
14. Gershkovich, A.A. and Kibirev, V.K., Khimicheskii sin-
tez peptidov (Chemical Synthesis of Peptides), Kiev:
Naukova Dumka, 1992.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 2 2006