Cytosol-mimetic chemistry: Kinetics of the trypsin-catalyzed hydrolysis of p-nitrophenyl acetate upon addition of polyethylene glycol and N-tert-butyl acetoacetamide

Article abstract of DOI:10.1021/ja034298f

The sensitivity of an enzyme to its environment has provoked much interest both for its immediate relevance to biochemistry and for the use of enzymes in chemical synthesis. The intercellular or extracellular environment in which an enzyme naturally opera

Full text of DOI:10.1021/ja034298f

Published on Web 05/16/2003
Cytosol-Mimetic Chemistry: Kinetics of the Trypsin-Catalyzed Hydrolysis of
p-Nitrophenyl Acetate upon Addition of Polyethylene Glycol and N-tert-Butyl
Acetoacetamide
Nabil Asaad* and Jan B. F. N. Engberts*
Physical Organic Chemistry Unit, Stratingh Institute, UniVersity of Groningen, Nijenborgh 4,
9747 AG Groningen, The Netherlands
Received January 23, 2003; E-mail: j.b.f.n.engberts@chem.rug.nl
The sensitivity of an enzyme to its environment has provoked
much interest both for its immediate relevance to biochemistry and
for its importance in the use of enzymes in chemical synthesis.1
This study examines the effect of concentrated solutions of
biomimetic compounds on enzyme reactivity.
The cytosol is an aqueous region, surrounded by a lipid bilayer
membrane, crowded with macromolecules and solutes totaling
several hundred grams per liter.2 This environment is markedly
different from the dilute aqueous solutions generally used as
background comparisons for biochemical processes.3 Indeed, in a
recent book Ball asks: “What is the structure of water in the cell?
This is one of the most important unresolVed issues in biology.”4
In the cellular environment the rate of diffusion, especially of large
molecules, is reduced5 and macromolecular crowding agents
promote protein folding and self-association.6 Our aim is to study
biologically relevant chemistry in biologically relevant medias
Figure 1. Eadie-Hofstee plots for the trypsin-catalyzed hydrolysis of
pNPOAc at varying concentrations of PEG (Mw 8000), 25 °C, and pH 7.7.
ultimately to mimic the environment found inside cells and to study
its properties as a chemical medium.
It would be reasonable to assume that as long as the active site
of an enzyme is shielded from the bulk solution, the catalytic rate
constant (kcat) would be unaffected by the addition of cosolutes or
crowding agents, up until they cause denaturation or affect the
diffusional rate-limit. The substrate binding constant (KM) would
be expected to be sensitive to solvent composition since it depends
on the chemical potential of the substrate free in solution. Substrate
binding also requires the dehydration of both the active site and
substrate, hence might be dependent on water activity. Previous
studies have highlighted the importance of water concentration,7
water activity and cosolute nature,8 and dielectric constant with
simultaneous competitive inhibition;9 however, no clear picture has
emerged.
We selected the trypsin-catalyzed hydrolysis of p-nitrophenyl
acetate (pNPOAc) as a model enzyme reaction which can conve-
niently be followed by UV-visible spectroscopy.10 Our initial
choice of crowding agent and cytosol mimic was bovine serum
albumin (BSA). However, we observed catalysis of the ester
hydrolysis by BSA.11 We therefore changed the additive to inert
poly(ethylene glycol) (PEG, Mw 8000) which has been widely
employed as a solubilizing agent and stabilizer in chemoenzymatic
synthesis.12 An additional solute, N-tert-butyl acetoacetamide
(NBAA) was studied to assess whether the observed kinetics were
a property of the PEG additive or a general response of the enzyme
to changes in its environment. NBAA is chemically inert in our
test system and contains the amide linkage (which is ubiquitous
inside the cell) and a balance of polar and hydrophobic groups
reflecting those found in biomolecules. As a relatively small
molecule, it would also not be expected to produce the same effects
on diffusion and association as PEGs.
Trypsin catalysis was measured for a series of solutions ranging
from no added solute up to 395 g L-1 of PEG and up to 269 g L-1
of NBAA.13 Monitoring the concentration of the product rather than
the substrate allowed the use of four convenient initial substrate
concentrations in the region of KM, at the expense of having to
calibrate against the extinction coefficient of p-nitrophenolate in
each PEG and NBAA solution. Kinetic parameters kcat and KM were
determined by the Eadie-Hofstee method (Figure 1).14
The observed Eadie-Hofstee plots are of the form classically
expected for noncompetitive inhibition of an enzyme.15 Substrate
binding is unaffected (mean KM ) 5.57 × 10-4 M); hence, PEG
does not compete for the active site and does not distort its structure.
The added PEG does however cause a decrease in kcat from 3.85
× 10-3 s-1 to 1.44 × 10-3 s-1 over the PEG concentration range
studied. Interestingly, the decline in the catalytic rate constant is
directly proportional to the concentration of added PEG (which
varies linearly with the concentration of water) and the same linear
dependence of kcat on PEG concentration is observed when PEG
(Mw 1500) is used (not shown). The same profile was obtained for
NBAA, but with an even greater decline in kcat (Figure 2). To allow
for a fair comparison of the differently sized solutes, kinetic
parameters are plotted as a function of molar water concentration,
rather than solute concentration.
Addition of simple solutes to water affects both polarity and water
activity. Polarity, as measured by Reichardt’s solvatochromic ET-
(30) probe,16 varies linearly with the concentrations of solute and
water in binary mixtures. Hence, a property that varies linearly with
water concentration may indicate a dependence on polarity. Water
activity, as measured by vapor pressure osmometry, is nonlinear
with concentration (Figure 3).
9
6874
J. AM. CHEM. SOC. 2003, 125, 6874-6875
10.1021/ja034298f CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
In summary, the presence of large concentrations of inert solutes
has only a modest effect on enzyme activitysless than an order of
magnitude reduction in kcat. Apparently, the catalytic process hardly
reacts to the large reduction in the concentration of water in the
model system. The enzyme probably retains its specific hydration
state, and consequently it is difficult to pinpoint the exact reasons
for the small reduction in kcat. It is noteworthy that the decline in
kcat is linear with increasing additive concentration. Comparison to
solvent polarity data and the nucleophilic reaction of the substrate
with TRIS indicate that this appears to be a medium effect on
enzyme chemistry and further attention should be paid to environ-
mental effects upon enzyme catalysis in vivo.
Acknowledgment. We are grateful to the NRSCC for financial
Figure 2. Variation of enzyme kinetic parameters with solute concentration
at 25 °C and pH 7.7. The solid lines are linear fits to kcat
support of this work.
.
Supporting Information Available: Eadie-Hofstee plots for
trypsin-catalyzed pNPOAc hydrolysis in the presence of NBAA. Plots
of pseudo-first-order rate constant against TRIS concentration for buffer-
catalyzed pNPOAc hydrolysis at different PEG concentrations (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Fersht, A. Enzyme Structure and Mechanism, 2nd ed.; Freeman: New
York, 1985. (b) Klibanov, A. M. Trends Biotechnol. 1997, 15, 97-101.
(c) Mesiano, A. J.; Beckman, E. J.; Russel, A. J. Chem. ReV. 1999, 99,
623-633.
(2) This corresponds to a water concentration in the region of 30-40 M. (a)
Cayley, S.; Lewis, B. A.; Guttman, H. J.; Record, M. T., Jr. J. Mol. Biol.
1991, 222, 281-300. (b) Neihardt, F. C.; Umbarger, H. E. Chemical
Composition of Escherischia coli. In Escherischia coli and Salmonella
typhimurium: Cellular and Molecular Biology; American Society for
Microbiology: Washington, DC, 1987.
(3) Ellis, R. J. Trends Biochem. Sci. 2001, 26, 596-604.
(4) Ball, P. H2O: A Biography of Water; Phoenix: London, 2000.
(5) Verkman, A. S. Trends Biochem. Sci. 2002, 27, 27-33.
(6) Morar, A. S.; Wang, X.; Pielak G. J. Biochemistry 2001, 40, 281-285.
(b) Rivas, G.; Fernandez, J. A.; Minton, A. P. Biochemistry 1999, 38,
9379-9388.
(7) Dzingeleski, G. D.; Wolfenden, R. Biochemistry 1993, 32, 9143-9147.
(8) Bell, G.; Janssen, A. E. M.; Halling, P. J. Enzyme Microb. Technol. 1997,
20, 471-477.
(9) Mares-Guia, M.; Figueirdo, A. F. S. Biochemistry 1972, 11, 2091-2098.
(10) It should be noted that trypsin is an extracellular enzyme that operates in
similarly concentrated media.
(11) Analogous catalysis of the Kemp elimination has been observed: Holl-
felder, F.; Kirby, A. J.; Tawfik, D. S. Nature 1996, 383, 60-63.
(12) Matsushima, A.; Kodera, Y.; Hiroto, M.; Nishimura, H.; Inada, Y. J. Mol.
Catal. B: Enzymol. 1996, 2, 1-17.
(13) Initial rates were obtained by linear regression of the first 2000 s of
absorbance/time data converted to p-nitrophenolate concentration using
the extinction coefficient of p-nitrophenol at the wavelength of maximum
absorption of the anion measured independently of the kinetic run in the
same buffer solution. Sample solutions containing the indicated solute
concentration and 7 µM trypsin, 40 mM TRIS buffer (pH 7.7), and 13
mM Ca2+ were preheated to 25 °C; to generate each data point 20, 30,
50, or 70 µL of p-nitrophenyl acetate stock solution in MeCN was then
injected.
Figure 3. Dependence of ET(30) and water activity17 on solute concentra-
tion at 25 °C.
It is inconceivable that PEG and NBAA have a common
noncompetitive binding site on the enzyme and unlikely that binding
of PEG and NBAA by trypsin at different sites remote from the
catalytic center accounts for the observed kinetics. A crowding-
induced change in enzyme conformation has previously been
proposed to account for lowering of enzyme activity,18 but unlike
PEG, NBAA is not a crowding agent. The observed kinetics must
therefore be attributable to an environmental effect on the enzyme,
the substrate, or both.
It is possible that the effect of the solute is limited to a
perturbation of the pKa of one or more catalytically important groups
on the enzyme. However, a study of the background nucleophilic
attack of TRIS on the substrate in the absence of enzyme19 revealed
an analogous linear variation of the (second-order) rate constant
with PEG concentration (not shown). Since isotopic substitution
experiments have shown that the solution phase nucleophilic and
chymotrypsin-catalyzed hydrolysis of pNPOAc proceed via ef-
fectively the same mechanism,20 we propose that in this system
the effect of the additive on solvent polarity is the dominant factor.
It appears therefore that the same effect of solvent polarity is
observed in both the enzyme-catalyzed and the solution-phase
reactions. This is reasonable since the active site of trypsin is
relatively exposed to solvent.21 The results presented here point to
a correlation between kcat and ET(30); however, there is insufficient
data to propose a direct link. We have observed similar trends in
a Diels-Alder reaction and an A1 acetal hydrolysis.22
(14) Hofstee, B. H. J. Nature 1959, 184, 1296-1298.
(15) Fersht, A. Structure and Mechanism in Protein Science; Freeman: New
York, 1998.
(16) This probe is sensitive to both the polarity of the solvent system and its
hydrogen-bond donating character. Reichardt, C. Chem. ReV. 1994, 94,
2319-2358.
(17) Water activity was measured in an AquaLab Series 3 vapor pressure
osmometer. Curve fit is based on a modified Flory-Huggins equation.
(18) Lonhienne, T. G. A.; Winzor, D. Biochemistry 2001, 40, 9616-9622.
(19) The rate of the background TRIS-catalyzed reaction is small relative to
that of the enzyme-catalyzed hydrolysis.
(20) Hess, R. A.; Hengge, A. C.; Cleland, W. W. J. Am. Chem. Soc. 1998,
120, 2703-2709.
(21) Nakasako, M. J. Mol. Biol. 1999, 289, 547-564.
(22) Asaad, N.; Engberts, J. B. F. N.; den Otter, M. J. Manuscript in preparation.
JA034298F
9
J. AM. CHEM. SOC. VOL. 125, NO. 23, 2003 6875