Interaction of α-Chymotrypsin with Dimethyl Sulfoxide: A Change of Substrate Could "Change" the Interaction Mechanism

Article abstract of DOI:10.1023/A:1026097308513

The kinetic behavior of α-chymotrypsin was studied in water-DMSO mixtures at concentrations of the organic solvent that do not cause irreversible denaturation of the enzyme. Various substrates (N-substituted derivatives of L-tyrosine) were found to displa

Full text of DOI:10.1023/A:1026097308513

Russian Journal of Bioorganic Chemistry, Vol. 29, No. 5, 2003, pp. 434–440. Translated from Bioorganicheskaya Khimiya, Vol. 29, No. 5, 2003, pp. 479–485.
Original Russian Text Copyright © 2003 by Eremeev.
Interaction of a-Chymotrypsin with Dimethyl Sulfoxide:
A Change of Substrate Could “Change”
the Interaction Mechanism
1
N. L. Eremeev
Faculty of Chemistry, Moscow State University, Vorob’evy gory, Moscow, 119899 Russia
Received September 19, 2002; in final form, November 20, 2002
Abstract—The kinetic behavior of α-chymotrypsin was studied in water–DMSO mixtures at concentrations
of the organic solvent that do not cause irreversible denaturation of the enzyme. Various substrates (N-substi-
tuted derivatives of L-tyrosine) were found to display substantially different kinetic patterns of interaction with
α-chymotrypsin, which can be described by totally different kinetic schemes. The differences were ascribed to
competition between the N-acyl group of the substrate and the DMSO molecule at the S2 site of substrate bind-
ing to the active site of the enzyme.
Key words: α-chymotrypsin in water–DMSO mixtures, mechanism of interaction with DMSO
1
INTRODUCTION
RESULTS AND DISCUSSION
Levashov et al. [1, 2] formulated the theoretical
principles of interactions between proteins and water-
miscible organic solvents. According to the mechanism
proposed by these authors, organic solvent molecules
substitute for water in the hydration shell of protein
globule when their concentration in the mixture
increases. The protein is irreversibly denatured when
the substitution degree reaches a critical value [3]. At
subcritical concentrations of organic solvent, the con-
formational changes induced in the protein globule by
the solvent are reversible [1, 4]. The critical concentra-
tion of a particular organic solvent for a concrete pro-
tein depends on the character of this pair: according to
spectral measurements, it is 4.2 M for the α-chymo-
trypsin–DMSO pair [5].
Study of the function of enzymes (in particular,
serine proteases) in water–organic mixtures is a rather
topical problem of fundamental and applied enzymol-
2
ogy. Traditionally, this problem is solved from the
dependence of the experimentally determined con-
stants of enzymatic reaction (catalytic and Michaelis
constants) on the concentration of organic solvent in
mixture. These dependences are used to conclude what
is the mechanism of the effector (in our case, organic
solvent) influence on the functioning of enzyme. The
simplicity of this approach is only seeming, because the
interpretation of the experimental data entails rather
serious problems. The physical sense of the mechanism
proposed by a researcher to explain an influence of
effector on the enzyme functioning strongly depends on
the researcher’s choice of a particular kinetic scheme.
However, this choice is primarily dictated by
researcher’s previous experience and his preference to
certain kinetic mechanisms.
The above-described mechanism presumes nonspe-
cific interactions between the protein and the organic
solvent. Therefore, one can expect that, at subcritical
concentrations, DMSO would slightly affect the cata-
lytic parameters of α-chymotrypsin. In fact, the cata-
lytic and the Michaelis constants are practically inde-
pendent on the DMSO concentration upon the BTNA
hydrolysis (Fig. 1). A small increase in the catalytic
constant can be explained either by the experimental
error or by a decreased energy of the substrate sorption
in the enzymic active site at increased permittivity of
medium, as done in [6].
The goal of present paper is the direct demonstration
of the fact that even such a parameter of the enzymatic
reaction as substrate may crucially change the kinetic
behavior of enzyme in water–organic mixtures. More-
over, various kinetic schemes may be used for the inter-
pretation of results obtained with the same substrate.
This statement is demonstrated by the example of the
well-studied system—α-chymotrypsin in the water–
DMSO mixtures.
At the same time, the observed picture is dramati-
cally changed in the case of another substrate, ATEE
(Fig. 2). The catalytic constant is monotonically
decreased, whereas the Michaelis constant slowly
increases up to 2 M DMSO concentration and then
increases rather strongly. Therefore, the experimental
1
Phone: +7 (095) 939-3417; e-mail: eremeev@enzyme.chem.
msu.ru
2
Abbreviations: ATEE, N-acetyl-L-tyrosine ethyl ester; ATNA, N-
acetyl-L-tyrosine p-nitroanilide; BTEE, N-benzoyl-L-tyrosine
ethyl ester; and BTNA, N-benzoyl-L-tyrosine p-nitroanilide.
1068-1620/03/2905-0434$25.00 © 2003 MAIK “Nauka /Interperiodica”

INTERACTION OF α-CHYMOTRYPSIN WITH DIMETHYL SULFOXIDE
kcat, s–1
435
kcat, s–1
250
2.6
(‡)
(‡)
200
150
100
50
2.4
2.2
2.0
1.8
1.6
0
Km, mM
0.20
Km, mM
(b)
(b)
5
0.18
4
3
2
1
0.16
0.14
0.12
0
1
2
3
4
[DMSO], M
1
2
3
4
0
[DMSO], M
Fig. 1. The dependence of the (a) catalytic and (b) Michaelis
constants for BTNA hydrolysis by α-chymotrypsin on the
DMSO concentration in mixture.
Fig. 2. The dependence of the (a) catalytic and (b) Michaelis
constants for ATEE hydrolysis by α-chymotrypsin on the
DMSO concentration in mixture.
data can be described by the scheme of noncompetitive acetyl-L-tyrosine [9–11] and N-acetyl-L-tryptophan
inhibition within the range of 0 to 2 M DMSO concen-
trations (Scheme 1). However, the inhibition scheme of
a mixed type [7] is more applicable in the whole range
of the DMSO concentrations (Scheme 2).
[12]. Such a discrepancy is commonly explained in
terms of the three-stage scheme of the reactions cata-
lyzed by the serine proteases (Scheme 3).
In addition, the rate-limiting stage of the reaction
with nitroanilide substrates is acylation of α-chymo-
trypsin (k2 Ӷ k3, kcat ≈ k2), i.e., the stage at which water
seems not to participate. In the case of ester substrates,
the rate-limiting stage is usually the deacylation of acyl
enzyme (k3 < k2) under the action of water molecule.
The water concentration decreases with the increase in
the content of organic solvent. However, it is difficult to
imagine how the decrease in water concentration by
10% could result in the twofold decrease in the catalytic
constant (cf. Fig. 2: the water concentration is as high
Thus, the first observed difference in the kinetic
behavior of α-chymotrypsin in the water–DMSO mix-
tures consists in the basically different dependences of
catalytic constant on the concentration of organic sol-
vent for two substrates, BTNA and ATEE. Note that
similar regularities have earlier been described. So, a
threshold character of the enzymatic activity was
noticed when p-nitroanilides of N-benzoyl-L-tyrosine
[1, 5, 7] and N-succinyl- and N-glutaryl-L-phenylala-
nine [8] were used as substrates. On the other hand, a
monotonic decrease in the catalytic constant was usu-
ally reported for the ester substrates, ethyl esters of N- as 50 M at the DMSO concentration of 1.2 M!).
KS
KS
kcat
kcat
E + S
ES
E + ΣP
E + S
ES
E + ΣP
I
Ki
I
KiE
I
KiES
IE
IE
IES
Scheme 2.
Scheme 1.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 5 2003

436
EREMEEV
Table 1. Constants of separate stages of the ATEE hydrolysis by α-chymotrypsin obtained according to the method of the
additional nucleophil (Schemes 4, 5)
Organic solvent
DMSO
kcat, s–1
k2, s–1
k3, s–1
KS, mM
205 ± 64
192
4600 ± 600
5000
238 ± 85
200
13.0 ± 1.0
17.2
1,4-Butanediol [14]
Acetoxime [15]
Ethanol [16]
130
3380
135.2
16.0 ± 0.4
5.8 ± 0.5
219 ± 11
3240 ± 1420
238 ± 3
Table 2. Constants of separate stages of hydrolysis by α-chymotrypsin of the substrates under study
Substrate
kcat, s–1
k2, s–1
k3, s–1
ATEE*
BTEE*
BTNA
ATNA
192
85.9
5000
249
200
131
2
200**
131**
0.04
* Data from [17].
** The value of k is assumed to be the same as for the corresponding acyl-chymotrypsin.
3
The above-described situation is quite clear for an of this method is also suitable for the determination of
individual rate constants of the three-stage reaction if
the reversible complex of acyl-enzyme with the effector
I is selectively formed (Scheme 5). In this case, the cal-
culated value of k4 in Scheme 4 reflects the concentra-
tion ratio of the productive acyl-enzyme and the non-
productive acyl-enzyme–effector complex. By apply-
ing the method of an additional nucleophil to the
experimental data (Fig. 2), we obtained constants of the
individual stages in the hydrolysis of ATEE by α-chy-
motrypsin (Table 1). A good agreement of our results
with literature data testifies to the efficiency of such
approach.
organic solvent that, at the stage of the acyl enzyme
deacylation, can act as an additional nucleophil com-
peting with water for the formation of the reaction
product P2, measured experimentally [14] (Scheme 4).
Berezin et al. [14, 15] developed in the early 1970s
the method based on the Scheme 4 for the calculation
of individual rate constants in the hydrolysis of sub-
strates by the serine proteases with use of alcohols and
oximes. DMSO is chemically inert toward the hydroly-
sis of acyl-chymotrypsin. However, calculations of the
kinetic schemes show that the mathematical apparatus
Now, at the first sight, the situation is rather clear:
organic solvent slows down (by one or another mecha-
nism) the stage of enzyme deacylation and does not
affect the acylation stage. As a result, at the hydrolysis
of the nitroanilide substrates by α-chymotrypsin (k2 Ӷ
k3, kcat = k2), the catalytic constant should slightly
depend (or should not depend at all) on the concentra-
tion of organic solvent. At the same time, an increase in
the solvent concentration in the mixture should result in
a monotonous reduction of this parameter at the hydrol-
ysis of ester substrates (k3 < k2) (cf. Figs. 1, 2). The real-
ity, however, entirely disproves this supposition. Let us
consider the BTEE hydrolysis at which k3 is lower than
k2 (Table 2) but not by an order of magnitude as for
ATEE but only approximately twofold (Fig. 3). As seen
from the figure, here the catalytic constant does not
decrease along with the increase in the DMSO concen-
tration, as would be expected for ester substrates.
Instead, it is practically independent on the solvent con-
centration, which resembles in appearance the similar
pattern observed in the case of BTNA hydrolysis
(Fig. 1).
KS
k2
k3
E + S
ES
EA
E + P2
P1
Scheme 3.
E + P2
KS
k2
E + S
ES
EA
P1
E + P3
Scheme 4.
KS
k2
k3
E + S
ES
EA
E + P2
I
I, Ki
P1
IEA
Scheme 5.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 5 2003

INTERACTION OF α-CHYMOTRYPSIN WITH DIMETHYL SULFOXIDE
kcat, s–1
437
kcat, s–1
(‡)
(‡)
90
80
70
0.08
0.06
0.04
0
Km, mM
0.08
Km, mM
1.0
(b)
(b)
0.8
0.6
0.4
0.2
0.07
0.06
0.05
0.04
0
1
2
3
4
1
2
3
4
0
[DMSO], M
[DMSO], M
Fig. 3. The dependence of the (a) catalytic and (b) Michaelis
constants for BTEE hydrolysis by α-chymotrypsin on the
DMSO concentration in mixture.
Fig. 4. The dependence of the (a) catalytic and (b) Michaelis
constants for ATNA hydrolysis by α-chymotrypsin on the
DMSO concentration in mixture.
The dependence of the catalytic constant of ATNA
hydrolysis on the DMSO concentration (Fig.4) brings
in the final verdict to the role of the nature of hydrolyz-
able bond as a factor that controls the rate-limiting
stage of the substrate hydrolysis and, accordingly, the
appearance of the dependence of this constant on the
concentration of organic solvent. No speculations on
the differences between the ester and nitroanilide sub-
strates of α-chymotrypsin can explain twofold activa-
tion effect that is observed at 1 M DMSO concentra-
tion. At the same time, the absolutely different kinetic
schemes are applicable, which depends on the used
DMSO concentrations. The experimental data obtained
at up to 1 M organic solvent concentrations can be
described by means of the scheme of noncompetitive
activation (Scheme 6), whereas the basically different
scheme is necessary for the data obtained at broader
range of DMSO concentrations. It is conventional to
describe the bell-shaped dependences of this kind
through a successive addition of two effector mole-
cules, the first of which activates the enzyme and the
other slows down the hydrolysis rate (Scheme 7; for the
classical example, see the pH dependence for α-chy-
motrypsin).
In such a manner, minimal structural changes in the
substrate molecule (Table 2) result in dramatic changes
in the kinetic behavior of α-chymotrypsin in water–
DMSO mixtures. In fact, schemes 1–7 practically
reflect the whole range of known kinetic manifestations
of the interactions of reversible effectors with biocata-
lysts [18]. How can we explain such a behavior of the
enzyme?
Let us analyze the summarized data on the changes
of relative catalytic constants for the hydrolysis of var-
ious substrates by native α-chymotrypsin in depen-
dence on the DMSO concentration in mixture (Fig. 5).
One can see that the structure of N-substituent of the L-
tyrosine amino group is the determining factor. The
KS
kcat
E + S
ES
E + ΣP
A
KA
A
αKA
βkcat
αKS
AE + S
AES
AE + ΣP
here α = β > 1.
Scheme 6.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 5 2003

438
EREMEEV
kcat/kcat(water)
S2
S1
S1'
organic solvent
2.5
2.0
1.5
1.0
0.5
1
2
3
4
L-Tyr
Ac
L-Tyr
X
Bz
X
Ac
0
1
2
3
4
[DMSO], å
L-Tyr
X
Bz
S2
L-Tyr
X
S1
S1'
S2
S1
S1'
Fig. 5. The dependence of the relative catalytic constants for
the (1) ATEE, (2) BTEE, (3) BTNA, and (4) ATNA hydrol-
ysis by α-chymotrypsin on the DMSO concentration in
mixture.
Fig. 6. The hypothetical scheme of DMSO interaction with
the active site of α-chymotrypsin.
kinetic data on the hydrolysis of the ester and nitroanil- one can suppose that the N-benzoyl substituent dis-
ide N-benzoyl-substituted substrates are practically places this molecule at the stage of formation of the
identical, whereas the replacement of the N-benzoyl enzyme–substrate complex; i.e., the structures of the
substituent by acetyl group results in dramatic differ- enzyme–substrate complexes formed in water and
ences in the observed kinetic picture: the activity drops water–DMSO mixtures are identical. At the same time,
in the case of ester substrate and is significantly acti- such a displacement is energetically unfavorable in the
vated in the case of nitroanilide substrate at the same case of the significantly less hydrophobic acetyl group.
DMSO concentrations. Evidently, such a difference is a Consequently, in the water–DMSO mixtures, α-chy-
result of a specific interaction of DMSO with the pro- motrypsin catalyses the hydrolysis of N-substituted
tein globule in a region of the sorption site of the sub- substrates in the native structure of the enzyme–sub-
strate N-acyl group (S2-site according to the Shechter– strate complex, whereas the structure of this complex is
changed when N-acetyl substrates are used due to the
interaction of protein globule with DMSO molecules
(Fig. 6).
Berger terminology [19]).
The binding at S2-site is known to occur due to the
hydrophobic interactions with Ile99 and/or Trp215 res-
idues [20]. In this case, the substrates with more hydro-
phobic N-benzoyl substituent are bound to chymo-
trypsin substantially more efficiently: a value of the
standard free energy of the enzyme–substrate complex
decreases by 2.7–2.9 kcal/mol upon the transition from
N-acetyl to N-benzoyl amino acid derivatives [21].
Therefore, if DMSO molecule could occupy S2-site,
The protective effect of N-benzoyl group of sub-
strate on the active site of α-chymotrypsin becomes
especially evident in water–DMSO mixtures at high
temperatures (Fig. 7). One can see that the DMSO con-
centration corresponding to the maximal catalytic
activity of enzyme is shifted from 1.5 to 0.3 M in the
case of ATNA hydrolysis and the temperature increase
from 20 to 30°ë. At the same time, the increased tem-
perature does not affect the dependence of the catalytic
constant on the DMSO concentration in the case of
BTEE hydrolysis; an increase in the DMSO concentra-
tion also does not affect the temperature dependence of
this parameter. These results support the above pre-
sumption that the cause of the kinetic effects observed
upon the hydrolysis of N-substituted substrates is a con-
formational change in the active site and a preferable
binding of the substrate N-benzoyl group to S2-site of the
active site of α-chymotrypsin rather than the molecule of
organic solvent. Amino acid residues become more
mobile at higher temperatures, which facilitates confor-
mational changes in the active site of α-chymotrypsin
KS
kcat
E + S
ES
E + ΣP
A
KA
A
"
KA
'
KS
αkcat
AE + S
AES
AE + ΣP
'
A
KA
A
'''
KA
"
KS
βkcat
A2E + S
A2ES
A2E + ΣP
here α > 1, ‡ β < α.
Scheme 7.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 5 2003

INTERACTION OF α-CHYMOTRYPSIN WITH DIMETHYL SULFOXIDE
439
behavior of α-chymotrypsin in water–DMSO mixtures.
Note that DMSO is not a unique solvent in respect of
the kinetic properties of α-chymotrypsin; similar
effects have been also demonstrated for water–ethanol
mixtures [16]. This means that, in some cases, organic
solvent is an effector of a particular enzymatic reaction
rather than an effector of the enzyme itself. This implies
that a great caution is necessary when considering the
regularities of the enzyme behavior established in
water–organic mixtures with the use of a model sub-
strate. A thoughtless application of such regularities to
another reaction (change of substrate) may result in cat-
astrophic mistakes.
(a)
kcat, s–1
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0
28
26
24
22
20
EXPERIMENTAL
3
Crystalline α-chymotrypsin from bovine pancreas
(grade A) was from OAO Samson (St. Petersburg, Rus-
sia); N-acetyl-L-tyrosine ethyl ester, N-benzoyl-L-
tyrosine p-nitroanilide, and N-benzoyl-L-tyrosine ethyl
ester were from Sigma (United States); and DMSO was
from OAO Khimreaktivkomplekt (Russia). N-Acetyl-
L-tyrosine p-nitroanilide was synthesized according to
the procedure described in [22].
2
1
[DMSO], å
0
(b)
kcat, s–1
450
400
350
300
Salts and buffer components (analytical grade) were
from Reakhim (Russia).
250
200
150
100
50
The activity of the wild-type a-chymotrypsin
toward ester substrates ATEE and BTEE was deter-
mined using the initial rates of hydrolysis in a 0.2 M
NaCl + 0.02 M CaCl2 solution at 20°ë and pH 8.0 by
potentiometric titration on an RTA 822 pH-stat (Radi-
ometer, Denmark).
32
28
The activity of the wild-type a-chymotrypsin
toward nitroanilide substrates. ATNA and BTNA
was determined using the initial rates of hydrolysis in
0.05 M sodium phosphate buffer, pH 8.0, at 20°ë on
an Hitachi 124 spectrophotometer at the wavelength
of 390 nm. Molar extinction coefficients of the prod-
uct (p-nitroaniline) were determined in independent
experiments for each used concentration of organic
solvent.
3
24
2
[DMSO], å
1
20
0
Fig. 7. The dependence of the catalytic constants of
(a) ATNA and (b) BTEE hydrolysis by α-chymotrypsin on
the temperature and the DMSO concentration in the mix-
ture.
Parameters of the Michaelis–Menten equation
were calculated from the dependences of the initial
rates of hydrolysis on the substrate concentration in the
Lineweaver–Burk double reciprocal coordinates.
upon the DMSO binding to S2-site. This is kinetically
appeared as a shift of the maximum of the catalytic
activity to lower concentrations of organic solvent in
the case of ATNA hydrolysis. In the case of BTEE
hydrolysis, the hydrophobic interactions of N-benzoyl
group with the amino acid residues of S2-site become
stronger, which results in stabilization of the structure
of the enzyme–substrate complex and in uncoupling of
ACKNOWLEDGMENTS
I thank V.V. Ryzhova and N.A. Uskova (Faculty of
the effects of temperature and DMSO concentration on Chemistry, Moscow State University) for the ATNA
the catalytic constant of the reaction.
synthesis.
Thus, as said above, minimal structural changes in
This work was partly supported by the Russian Foun-
substrate result in dramatic changes in the kinetic dation for Basic Research, project no. 98-03-32204a.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 5 2003

440
EREMEEV
11. Eremeev, N.L., Belyaeva, E.A., and Kazanskaya, N.F.,
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