Effect of crowding by dextrans on the hydrolysis of N-Succinyl-L-phenyl-Ala-p-nitroanilide catalyzed by α-chymotrypsin.

TLDR: The results indicate that the volume occupied by the crowding agent, but not its size, plays an important role in the rate of this reaction, with a v(max) decay and a K(m) increase obtained when the dextran concentration in the sample was increased.

Abstract: Traditionally, studies on the diffusion-controlled reaction of biological macromolecules have been carried out in dilute solutions (in vitro). However, in an intracellular environment (in vivo), there is a high concentration of macromolecules, which results in nonspecific interactions (macromolecular crowding). This affects the kinetics and thermodynamics of the reactions that occur in these systems. In this paper, we study the crowding effect of large macromolecules on the reaction rates of the hydrolysis of N-succinyl-L-phenyl-Ala-p-nitroanilide catalyzed by α-chymotrypsin, by adding dextrans of various molecular weights to the reaction solutions. The results indicate that the volume occupied by the crowding agent, but not its size, plays an important role in the rate of this reaction. A v(max) decay and a K(m) increase were obtained when the dextran concentration in the sample was increased. The increase in K(m) can be attributed to the slowing of protein diffusion, due to the presence of crowding. Whereas the decrease in v(max) could be explained by the effect of mixed inhibition by product, which is enhanced in crowded media. As far as we know, this is the first reported experiment on the crowding effect in an enzymatic reaction with a mixed inhibition by product.

Figures

Table 1. Kinetic constants of the hydrolysis of N-succinyl-L-phenyl-Ala-p-nitroanilide catalyzed by alpha-chymotrypsin for different concentrations of Dextran (in mg/mL). The value of the fitting parameter r 2 is also given.

Full text

1
Effect of crowding by Dextrans on the hydrolysis of N-succinyl-L-phenyl-Ala-p-
nitroanilide catalyzed by alpha-chymotrypsin
Isabel Pastor
†,
*, Eudald Vilaseca
†
, Sergio Madurga
†
, Josep Lluís Garcés
‡
, Marta
Cascante
§
and Francesc Mas
†
†
Department of Physical Chemistry and the Research Institute of Theoretical and
Computational Chemistry (IQTCUB) of the University of Barcelona (UB), C/ Martí i Franquès,
1, E-08028 Barcelona (Spain)
‡
Department of Chemistry, University of Lleida (UdL), Lleida (Spain)
§
Department of Biochemistry and Molecular Biology and the Institute of Biomedicine (IBUB)
of the University of Barcelona (UB) and IDIBAPS, Barcelona (Spain)
*
Corresponding author:
Isabel Pastor
E-mail:
i.pastor@ub.edu
Fax: (+34) 934021231

2
ABSTRACT
Traditionally, studies on the diffusion-controlled reaction of biological macromolecules
have been carried out in dilute solutions (in vitro). However, in an intracellular
environment (in vivo), there is a high concentration of macromolecules, which results in
non-specific interactions (macromolecular crowding). This affects the kinetics and
thermodynamics of the reactions that occur in these systems. In this paper, we study the
crowding effect of large macromolecules on the reaction rates of the hydrolysis of N-
succinyl-
L
-phenyl-Ala-p-nitroanilide catalyzed by alpha-chymotrypsin, by adding
Dextrans of various molecular weights to the reaction solutions. The results indicate that
the volume occupied by the crowding agent, but not its size, plays an important role in
the rate of this reaction. A v
max
decay and a K
m
increase were obtained when the Dextran
concentration in the sample was increased. The increase in K
m
can be attributed to the
slowing of protein diffusion, due to the presence of crowding. Whereas the decrease in
v
max
could be explained by the effect of mixed inhibition by product, which is enhanced
in crowded media. As far as we know, this is the first reported experiment on the
crowding effect in an enzymatic reaction with a mixed inhibition by product.
Keywords: enzyme kinetics, alpha-chymotrypsin, macromolecular crowding, mixed
inhibition by product

3
INTRODUCTION
The cell cytosol is an aqueous medium that is crowded with macromolecules and
solutes, which occupy up to 40% of its total volume
1
. However, studies of biochemical
processes have usually been performed in dilute solutions, in which the environment is
markedly different from the cytosol. In the cellular environment, the rate of diffusion is
reduced
2-8
, and can even be anomalous at short times. In addition, the macromolecular
crowding agent promotes processes such as protein folding, self-association and protein
binding
9-22
.
Hence, the presence of large concentrations of inert solutes can affect the
enzymatic activity.
However, quite a few studies have explored the effects of crowding on enzyme
catalysis, even in vitro. Laurent carried out one of the first studies on enzymatic
reactions in macromolecular crowded media in 1971
23
. He
studied enzyme reactions in
polymer media as an initial attempt to describe how the environment affects the
intracellular enzyme function. This was nevertheless a complete study, because Laurent
examined the effect of the polymer presence in several situations: the degradation of
hyaluronic acid by hyaluronate lyase in the presence of polyethylene glycol; the lactate
dehydrogenase reaction in Dextran (40 kDa) solution, and the cleavage of benzoyl
DL
-
arginine p-nitroanilide by trypsin and its inhibition by serum albumin in Dextran
solution. In all these cases, the presence of the polymer produced a moderate decrease in
the apparent Michaelis-Menten (MM) constant, , or in the apparent inhibition rate
constant, , in the case of inhibition. In 1981, Minton and Wilf
9
studied the effect of
macromolecular crowding on the various kinetic steps in the enzymatic processes
(enzyme [E] and substrate [S] meet and form the ES complex; the formation of an
enzyme-product complex from the ES complex; and the release of product from the

4
enzyme-product complex) of glyceraldehyde-3-phosphate dehydrogenase. They
predicted that the rate of an enzymatic reaction will decrease when there is an increase
in the concentration or the size of the crowding agent. In other words, the excluded
volume decreases both the MM constant, , and the catalytic constant, , when the
enzymatic reaction follows the Michaelis-Menten mechanism. However, most
subsequent studies reported that a high concentration of neutral polymers only had a
moderate influence on enzyme reactions. Briefly, a slight decrease in is frequently
found, regardless of the properties of the crowding agent
24-30
. However, the effect of the
crowding agent on is diverse: in some cases, increases
24, 28-32
, whereas in other
cases it decreases
9, 26-27, 32
. It should be noted that these studies were carried out in the
presence of a single kind of polymer (e.g. Dextrans or Ficoll) of a fixed size (usually
small).
The range of experimental conditions covered by these studies is not wide
enough to fully understand the phenomenology of enzymatic kinetics in crowded media.
In particular, two important aspects have to be investigated in depth. Firstly, to obtain a
more realistic description of the cell cytosol environment, a large variety of
macromolecules of different sizes and shapes should be considered. We should also take
into account that each type of macromolecule may have a different influence on the
enzymatic reaction. Secondly, when the diffusion process of proteins in macromolecular
crowded media is studied, it is very important to consider not only the volume occupied
by the crowding agent, but also how this volume is distributed. Diffusion has been
found to depend on both the concentration and the size of the obstacles
33-35
. Both
aspects must be studied to improve our understanding of enzymatic reactions in cell
cytosol.

5
In this paper, we studied how the kinetics of an in vitro enzymatic reaction was
affected by the concentration and the size of the crowding agents. We chose the alpha-
chymotrypsin hydrolysis of N-succinyl-L-phenyl-Ala-p-nitroanilide as a model
reaction, because there is only a minimal change in the excluded volume due to the
small size of substrates and products. Thus, we studied the effect of the excluded
volume in the reaction due to the presence of crowding agents. Macromolecular
crowding was mimicked using Dextrans from 5 to 410 kDa. There were three
advantages of using alpha-chymotrypsin protein in this study: the absence of known
interactions with Dextrans (crowding agents); the protein size (hydrodynamic radius, r
h
= 2.33 nm), which is intermediate between those of the selected crowding agents; and
the protein’s isoelectric point of 5.4, which implies that it was negatively charged in the
buffer that was used (pH 8). This reduces the risk of aggregation, which is higher in
crowded media. In this paper, we examine how this enzymatic reaction is affected by
the presence of Dextrans of different sizes at different concentrations. In particular, we
analyzed the effect of macromolecular crowding on the values of the and
parameters of this reaction.
MATERIALS AND METHODS
Chemicals
Alpha-chymotrypsin (E.C. 3.4.21.1) from bovine pancreas type II (60 Umg
-1
), which
was used without further purification, and N-succinyl-
L
-phenyl-Ala-p-nitroanilide were
purchased from Sigma-Aldrich Chemical (Milwaukee, WI, USA). Dextran (from
Leuconostoc mesenteroides) of molecular weight 5, 50, 150, 275 and 410 kDa was
purchased from Fluka (Buchs, Switzerland). The polydispersities of the Dextrans were

Frequently asked questions

Q1. What are the contributions in "Effect of crowding by dextrans on the hydrolysis of n-succinyl-l-phenyl-ala-p- nitroanilide catalyzed by alpha-chymotrypsin" ?

In this paper, the authors studied the effect of the presence of polyethylene glycol on enzyme reactions in a cell cytosol. 

Q2. What is the role of the macromolecular crowding agent in enzymecatalysis?

In addition, the macromolecular crowding agent promotes processes such as protein folding, self-association and protein binding 9-22 . 

Q3. What is the effect of crowding on the enzyme?

The decrease in with crowding could be ascribed to an increase inthe ratio of activity coefficients between the native enzyme and the enzyme-substrate complex, or to an increase in chemical activity of the small molecule substrate in highly non ideal crowded solution 26-28 . 

Q4. Why did the authors choose the alphachymotrypsin hydrolysis as a model?

The authors chose the alphachymotrypsin hydrolysis of N-succinyl-L-phenyl-Ala-p-nitroanilide as a model reaction, because there is only a minimal change in the excluded volume due to the small size of substrates and products. 

Q5. What is the effect of the crowding on the inhibition of Nsuccinyl-?

the authors consider that the hydrolysis of Nsuccinyl-L-phenyl-Ala-p-nitroanilide catalyzed by alpha-chymotrypsin in crowded media is at least dependent on diffusion in the crowded media and on effective inhibition by product, which is enhanced by the presence of these crowding agents. 

Q6. What is the effect of crowding on the inhibition constants?

The inhibition constants Kic and Kiu can also increase with crowding, due to the decrease in the two bimolecular diffusion-controlled rate constants k1 and k-3 (Eqs. 7 and 8). 

Q7. What was the concentration of Dextran in the reaction mixture?

The reaction mixture contained a fixed concentration of 8.5 10 -6 M of alpha-chymotrypsin, the concentration of substrate (N-succinyl-L-phenyl-Ala-pnitroanilide) varied between 5.4 10 -5 and 4.8 10 -4 M, and the concentration of Dextran varied between 0 and 450 mg/mL. 

Q8. How did Michaelis and Menten find out that the enzyme activity was largely solved?

The difficulty of following an enzymaticreaction was largely solved when Michaelis and Menten showed that, under certain conditions, e.g. an excess of substrate, enzyme activity could be studied by measuring the initial rate of product formation. 

Q9. What is the effect of medium viscosity on the hydrolysis of alpha-chy?

They examined the effect of medium viscosity on the hydrolysis of different p-nitroanilides and found that in the hydrolysis of N-succinyl-Lphenyl-Ala-p-nitroanilide the value of increased with the viscosity of the medium. 

Q10. What is the effect of crowding on enzyme activity?

In contrast, with small obstacles the effect of crowding is partially offset by enhancement of the enzyme activity owing to a caging effect, which results in a minor decrease in rate.