Polyethylene glycol effect on the transient and steady state phases of p-nitrophenyltrimethyl acetate hydrolysis catalyzed by α-chymotrypsin

Article abstract of DOI:10.4067/S0717-97072013000400033

The effect of polyethylene glycol (PEG) on the hydrolytic activity of ?-chymotrypsin has been studied using p-nitrophenyltrimethyl acetate (PNTMA) as substrate. This substrate requires long times to reach the steady state condition, allowing the measureme

Full text of DOI:10.4067/S0717-97072013000400033

J. Chil. Chem. Soc., 58, Nº 4 (2013)
POLYETHYLENE GLYCOL EFFECT ON THE TRANSIENT AND STEADY STATE PHASES OF
P-NITROPHENYLTRIMETHYL ACETATE HYDROLYSIS CATALYZED BY α-CHYMOTRYPSIN
CRISTIAN CALDERÓN* AND EDUARDO LISSI
Facultad de Química y Biología, Universidad de Santiago de Chile, USACH
This work is dedicated to the memory of Elsa Abuin and Guillermo Contreras
(Received: July 22, 2013 - Accepted: September 3, 2013)
ABSTRACT
The effect of polyethylene glycol (PEG) on the hydrolytic activity of α-chymotrypsin has been studied using p-nitrophenyltrimethyl acetate (PNTMA) as
substrate. This substrate requires long times to reach the steady state condition, allowing the measurement by conventional methods of the fast initial “burst” step
present on the biphasic catalytic behaviour exhibited by α-chymotrypsin.
The presence of PEG increases both the initial and steady state rates of the reaction. This increase was observed at all the substrate concentrations employed.
A formal Michaelis-Menten treatment under steady state conditions shows that KM remains almost constant over the range of PEG concentrations studied (up to
2
0% w/v), whereas kcat moderately increases with the increment in PEG concentration.
The results obtained in this work are interpreted in terms of the following mechanism:
Keywords: Enzyme kinetics, chymotrypsin, p-nitrophenyl trimethylacetate, poly-ethyleneglycol
A calculation of k , k and K , derived form this mechanism of
addition of Dextran, PEG and Carbopol upon the activity of urease, evidencing
2
3
s
α-chymotrypsin reveals that PEG influences mainly k2 and k , leaving Ks
for all the polymers studied a decrease on urease activity. The observed effect
was explained in terms of a viscosity effect that slows the reaction due to a
restricted diffusion of the reagents.
3
unmodified at the polymer concentrations considered. The observed effect of
PEG could be related to its influence upon the acyl-enzyme complex (ES′) with
almost 99% of the enzyme complexed over the range of PEG concentrations
employed.
α-Chymotrypsin (α-CT) is a water-soluble enzyme (25 kDa; pI = 8.3) that
catalyzes the hydrolysis of peptidic bonds in proteins, being able to act both
5
-8
upon amide and ester linkages . The activity of the enzyme is modified by
the presence of lipid/water interfaces and extensive studies have been reported
INTRODUCTION
9
-11
regarding the characteristics of the process in reverse micellar solutions and
1
2-15
Studies on the effect of macromolecules on the catalytic activity of
enzymes are of interest due to the fundamental role of these processes in living
organisms and their wide range of industrial applications. Studies of enzyme
activity of biological relevance are currently carried out in diluted solutions.
Such environments are different from those in which enzymes perform their
biological function, where normally are present a large variety of other
macromolecules that can modify the rate of the catalytic process.
surfactants solutions below and above their CMC . Regarding its behaviour
in the presence of macromolecules, it has been reported that the presence
of HSA decreases the rate of hydrolysis of N-glutaryl-L-phenylalanine
1
6
p-nitroanilide (GPNA) but correction of the Michaelis-Menten parameter
taking into account de decrease in free substrate, showed that both kcat and KM
remains almost independent of the presence of albumin, demonstrating that
the depletion of the substrate is the main factor influencing the activity of the
enzyme in this system. Studies regarding the behaviour of α-CT in the presence
The presence of macromolecules on the media can modulate the activity
of the enzyme in a rather complex fashion. However, three main phenomena
1
7
of polyethylene glycol has also been performed . It has been reported that the
presence of the polymer increases the reaction rate in the steady state stage of
the hydrolysis of p-nitrophenyl acetate catalysed by α-chymotrypsin.
(individually or combined) can explain the changes observed in the enzyme
activity: effect of the added macromolecule on the free substrate concentration,
enzyme macromolecule interactions and excluded volume effects.
Changes in free substrate concentration due to the presence of
macromolecules (i.e. transport proteins) is a factor worth to be considered
regarding its influence on enzymatic activity. The effect of the addition of
albumin on the acyl chain specificity of lipase towards a series of triacylglicerols
In the present work we present data obtained in the hydrolysis of
p-nitrophenyltrimethyl acetate catalysed by α-chymotrypsin. This substrate
shows the same behaviour as the commonly used p-nitrophenyl acetate but,
due to the presence of the bulky trimethylacetate group (which introduces a
steric hindrance that stabilizes the acyl-enzyme complex), PNTMA requires
1
of various acyl-chain lengths as substrates was evaluated by Wang and col. .
1
8
These authors concluded that the inhibitory effect of albumin on the lipase
catalysed hydrolysis of trihexanoylglycerol can be explained in terms of the
high affinity of albumin towards this substrate.
longer times to reach the steady state condition , allowing an evaluation of the
kinetics of the whole enzymatic process, particularly for the transient phase of
1
9
the α-chymotrypsin mechanism, by conventional procedures .
Another factor to be accounted for into the analysis of the effect of the
presence of macromolecules into enzymatic activity is related to the enzyme/
macromolecule interaction. Calderón et al. , on the study of the effect of human
MATERIALS AND METHODS
2
serum albumin (HSA) on the rate of hydrolysis of 4-methylumbelliferyl-β-D-
N-N´-N´´-β-D-glucoside catalyzed by egg hen white lysozyme, revealed that
Reagents and equipment
α-Chymotrypsin(α-CT,TypeII,frombovinepancreas),polyethyleneglycol
(PEG, nominal molecular weight 3,400) and p-nitrophenyltrimethylacetate
(PNTMA) from Sigma were used as received. Ultrapure water obtained from
a Barnstead easy pure II equipment was employed to prepare all the solutions.
All measurements were performed at 25°C in TRIS/HCl buffer (pH = 7.0).
Absorption spectra and absorbances were recorded with a Hewlett-Packard
8453 UV-Visible spectrometer.
the presence of albumin decreases kcat for the reaction leaving K unmodified.
M
The observed variation on the kinetic behaviour evidenced was explained in
terms of the occurrence of the interaction HSA/Lysozyme, mainly due to the
correspondence between the variation of the catalytic rate constant and the
decrease of the free enzyme concentration.
In complex media, high concentration of macromolecular agents such as
proteins, polysaccharides, synthetic polymers, etc. effects of excluded volume,
commonly known as molecular crowding effects, are evidenced. Assad and
Reaction rate measurements
3
Engberts , studied the kinetics of the hydrolysis of p-nitrophenyl acetate
The rate of PNTA hydrolysis catalyzed by α-CT, was measured at 25°C
in aqueous buffered solution and in PEG (up to 20 % w/v) solutions at pH =
7 (10 mM TRIS-HCL buffer). The reaction was followed by registering the
absorbance of p-nitrophenol (PNP, 440 nm, ε = 17,700 M cm ) released over
the course of the reaction as a function of time. The extinction coefficient of
(PNA) catalysed by trypsin in the presence of poly ethylene glycol (PEG).
The increase in PEG concentration causes a moderate decrease in kcat while K
M
-
1
-1
-1
remains constant over the range of PEG concentrations studied (up to 395 g L ;
4
MW = 8000). On a similar topic, Derham and Harding , studied the effect of the
e-mail: cristian.calderon@usach.cl
2053

J. Chil. Chem. Soc., 58, Nº 4 (2013)
PNP was independent of PEG concentration in the range of concentrations used
in the present work. The reaction was initiated by the addition of an aliquot of
a concentrated solution of α-CT to a solution containing a given concentration
of PNTMA in buffer (or PEG solution) in order to obtain a final enzyme
concentration equal to 10 µM.
RESULTS AND DISCUSSION
The time course for the release of p-nitrophenol in the hydrolysis of
p-nitrophenyltrymethyl acetate catalyzed by α-chymotrypsin in absence and in
the presence of polyethylene glycol is shown in Figure 1. Both in presence and
absence of polyethylene glycol, the data show the two distinguishable “steps”
1
8
characteristics of α-chymotrypsin catalyzed processes . A very fast initial rate
or “burst” step is observed, followed by a decay in the rate of p-nitrophenol
release until steady state conditions are achieved. The presence of polyethylene
glycol increases both the initial and steady state rates of the reaction. This
-5
increase was observed at all the substrate concentrations employed (2x10 to
-4
7
x10 M).
Figure 2. Dependence of kcat and KM with PEG concentration. kcat (■); KM
(○). Each data point represents the mean of three independent experiments.
It is mandatory then, to delve into some explanation for the observed
behavior of kcat and KM, to evaluate the effect of PEG on k , k and the
2
3
relationship k / k (more commonly expressed as K ). According to the
-1
1
S
mathematical analysis developed by Bender et al. (14) on the study of the
kinetic behavior of the hydrolysis of p-nitrophenyltrimethylacetate catalyzed
by α-chymotrypsin, the determination of the decay constant (b) from the curve
obtained by subtracting the experimental PNP vs. time curve from the steady
state straight line (Figure 3) allows the evaluation of k and K , based on the
Figure 1. Time course of p-Nitrophenol (PNP) formation in the hydrolysis
of p-nitrophenyl trymethyl acetate catalyzed by α-chymotrypsin. In absence of
PEG (■); 20 % p/v PEG (○). PNTA = 7 x 10 M.
2
S
hyperbolic dependence between b and the substrate concentration proposed by
the authors (Eq. 3).
-4
In order to obtain some insight into the origin of the effect exerted by
polyethylene glycol, a formal Michaelis-Menten treatment of the data obtained
was performed. The dependence of kcat and K with the polyethylene glycol
M
concentration is shown in Figure 2. For K , within the uncertainty of the data,
M
a small increase is observed, indicating a negligible or non-existent effect
of polyethylene glycol on the binding process that leads to the substrate/
enzyme complex formation. On the other hand, kcat shows a more significant
dependence with the polyethylene glycol percentage, leading to an increase of
1
7
kcat of almost 6 times from zero to 20% polyethylene glycol. Abuin et al. in
a study on the effect of polyethylene glycol on the hydrolysis of p-nitrophenyl
acetate catalyzed by α-chymotrypsin, reported similar dependences of K and
M
kcat. In this system, KM remains constant over the range of PEG concentrations
employed (up to 30 % p/v PEG) and kcat shows a much smaller increase with PEG
concentration than that found in the present work using p-nitrophenyltrymethyl
acetate as substrate.
The α-chymotrypsin catalytic mechanism is commonly represented by the
following scheme.
Where E is the free enzyme, ES the enzyme/substrate complex, ES′ the
acyl-enzyme intermediate, PNP stands for p-nitrophenol and P is the acetate
moiety.
It can be demonstrated that, for the proposed mechanism, kcat and KM can
be expressed according to equations 1 and 2:
2
Figure 3. Schematic depiction of the mathematical treatment for the
estimation of the parameter b. Experimental PNP vs. time plot (●); extrapolated
steady state straight line (Δ); decay curve obtained by subtraction of ● from Δ.
2
054

J. Chil. Chem. Soc., 58, Nº 4 (2013)
Figure 4 shows the treatment of the kinetic data according to Eq. 3 at
different PEG concentrations. A hyperbolic dependence between the decay
constant b and the substrate concentration was observed at PEG concentrations
up to 20 % w/w. At the highest concentration of PEG considered, a tenfold
K shows little variation with the polyethylene glycol concentration (Figure
S
6). This could explain the behavior exhibited by K . According to eq. 2, K
M
M
depends upon K and the k / k ratio (because of k ˃˃˃ k ). It can be seen that
S
3
2
2
3
KM remains relatively constant at all the polyethylene glycol concentrations
employed, mainly because K and the k /k ratio remain essentially constant.
-4
increase in the exponential decay constant (at 7 x 10 M of PNTMA). The
prevalence of the hyperbolic behavior allows an analysis of the dependence of
k and K forα-chymotrypsin.witth PEG concentration..
S
3
2
2
S
The data obtained for k is shown in Figure 5. The magnitude of k readily
2
2
increases with the increase of polyethylene glycol concentration, having a value
almost ten times larger at 20% polyethylene glycol than the one determined in
absence of polyethylene glycol. The presence of the polymer in the reaction
media seems to have a considerable effect on the rate of the “burst” step of
the α-chymotrypsin catalytic mechanism. The variation of k3 is similar to
that observed for k . This results from the fact that, according to eq. 1, being
cat
k much larger than k k turns out to be equal to k The values of k and k
2
3,
3
cat.
2
3
obtained allow to postulate that, in spite of the increase on the reaction rate of
both the “burst” step and the stationary-state, the effect of polyethylene glycol
on the activity of α-chymotrypsin observed has its origin on its influence on the
process of release of the second product (the acetate fragment) from the acyl-
enzyme complex (ES′).
Figure 6. Dependence of Ks with PEG concentration. Data represent the
mean of three independent experiments and was fitted according to a linear
regression.
Theeffectofpolyethyleneglycolonthehydrolysisofp-nitrophenyltrimethyl
acetate hydrolysis catalyzed by α-chymotrypsin could be related to its influence
upon the acyl-enzyme complex (ES′). Almost 99% of the enzyme is in the form
of ES′ over the range of PEG concentrations employed. This was estimated by
the determination of the initial reaction rates (V ) and the steady state rate (V )
0
ss
both extrapolated at infinite substrate concentration (Eq. 4)
being α the fraction of enzyme in the form of the acyl-enzyme complex.
Similar results are obtained by using the k /k ratio (Eq. 5)
3
2
Figure 4. Dependence of the decay constant (b) with
p-nitrophenyltrimethylacetate concentration. No PEG (■); 10% PEG (●);
1
5% PEG (▲); 20% PEG (▼). Experiments were performed by triplicate.
Nonlinear curve fit was performed according to eq. 3 using Origin (OriginLab
Corp.) software.
The presence of PEG on the reaction media seems to induce a decrease
on the stability of ES′ favouring at the same time the transition between ES
and ES′. This phenomena might be originated by conformational changes on
the enzyme due to its interaction with PEG molecules. In spite of the lack
of charge on PEG this interaction must involve a direct association between
the protein and the polymer since excluded volume effects are not relevant at
the polymer concentrations employed in this work. In fact, at the highest PEG
concentrations employed (20 %) only 17 % of the solution volume is occupied
by the polymer ensemble. Further studies are required in order to ascertain
these observations.
CONCLUSIONS
The increment of PEG concentration increases the rate of both the
transient and steady state phase of the hydrolysis of p-nitrophenyltrimethyl
acetate hydrolysis catalyzed by α-chymotrypsin. The influence of polyethylene
glycol is exerted mainly by inducing instability over the acyl-enzyme complex,
while favoring the transition between the enzyme-substrate complex and
the acyl-enzyme complex. The observed behavior seems to be related with
conformational changes on the enzyme due to direct interaction with PEG
molecules.
Figure 5. Dependence of k and k with PEG concentration. k (●); k
3
∆). Experiments were performed by triplicate. The solid line represent the
2
3
2
(
exponential fit of the data.
ACKNOWLEDGMENTS
This work has been supported by FONDECYT, Chile.
2
055

J. Chil. Chem. Soc., 58, Nº 4 (2013)
REFERENCES
9. Y. Miyake, T. Owari, F. Ishiga, M. Teramoto. J. Chem. Soc. Faraday
Trans. 90, 979 (1994).
1
.
J-S. Wang, X. Wen, J.T. Backman, P.J Neuvonen. J. Pharmacol. Exp.
Ther. 302:43 (2002).
10. F. M. Menger, K. Yamada. J. Am. Chem. Soc. 101, 6731 (1979).
11. L.F. Aguilar, E. Abuin, E. Lissi. Arch. Biochem. Biophys. 388, 231 (2001).
12. E. Abuin, E. Lissi, R. Duarte. Langmuir 19, 5374 (2003).
13. E. Abuin, E. Lissi, C. Calderón. J.Colloid Interf. Sci. 308, 573 (2007).
14. E. Abuin, E. Lissi, R. Duarte. J. Molec. Catal. 31, 83, (2004).
15. E. Abuin, E.Lissi. R. Duarte. J. Colloid Interf. Sci. 283, 539 (2005).
16. E. Abuin, E. Lissi, M. Ahumada. C. Calderón. Protein J. 30, 143 (2011).
17. E. Abuin, E. Lissi, R. Bridi. J. Chil. Chem. Soc. 56, 948 (2011).
18. M.L. Bender, F. Kèzdy, F. C. Wedler. J. Chem. Ed. 44, 84 (1967).
19. C.E. Mcdonalds, A.K. Balls. J. Biol. Chem. 227, 727 (1957).
2
3
4
5
.
.
.
.
C. Calderón, E. Abuin, E. Lissi, R. Montecinos, Protein J. 30:367 (2011).
N. Assad, J. B. F. N. Engberts, J. Am. Chem. Soc. 15, 6874 (2003).
B. K. Derham, J. J. Harding, Biochim. Biophys. Acta 1764, 1000 (2006).
N. Spetri, F. Alfani, M. Cantarella, F. D’Amico, E. Germani, G. Savelli. J.
Mol. Catal. 6, 99 (1999).
P. Viparelli, F. Alfani, M. Cantarella. Biochem. J. 344, 765 (1999).
F. Alfani, F. M. Cantarella, N. Spetri, R. Germani, G. Savelli. Appl.
Biochem. Biothechnol. 88, 1 (2000).
6
7
.
.
8
.
P. Viparelli,F. Allfani, M. Cantarella. J. Mol. Catal. B: 15, 1 (2001).
2
056