Enzyme Technology
Effect of solute partition on the kinetics of immobilised enzymes
The solution lying within a few molecular diameters (l
10 nm) from the surface of an immobilised enzyme will be influenced by both
the charge and hydrophobicity of the surface. Charges are always present on
the surface of immobilised enzyme particles due to the amphoteric nature of
enzymes. Where these positive and negative charges are not equally balanced,
the net charge on the surface exerts a considerable effect over the properties
of the microenvironment. This surface charge, easily produced by the use of
ion-exchange or similar charged matrices for the immobilisation, repels molecules
of similar charge while attracting those possessing opposite charge. The force
of attraction or repulsion due to this charge is significant over molecular
distances but decays rapidly with the square of the distance from the surface.
A partitioning of charged molecules (e.g., substrates and products) occurs between
the bulk solution and the microenvironment; molecules of opposite charge to
the immobilised enzyme surface being partitioned into the microenvironment,
whereas molecules possessing similar charge to the immobilised enzyme surface
are expelled, with equal effect, into the bulk solution. The solute partition
may be quantified by introduction of the electrostatic partition coefficient
(L) defined by,
(3.1)
where [C0n+] and [A0n-] represent each cation and
anion bulk concentration, [Cn+] and [An-] represent their
concentration within the microenvironment and n is the number of charges on each
ion. L has been found to vary within the range of about 0.01 to 100;
L being greater than unity for positively charged enzymic surfaces and
less than unity for negatively charged surfaces. The effect of partition on
positively and negatively charged molecules is equal but opposite. For a
positively charged support, cations are partitioned away from the
microenvironment whereas the concentration of anions is greater within this
volume compared with that in the bathing solution. j depends on the
density of charge on and within the immobilised enzyme particle. It is greatly
influenced by the ionic strength of the solution. At high ionic strength the
raised concentration of charged solute molecules counteracts the charge on the
particles, reducing the electrostatic forces and causing j to approach
unity. Assuming Michaelis-Menten kinetics, the rate of reaction catalysed by an
immobilised enzyme is given by equation 1.9 where the substrate concentration is
the concentration within the microenvironment.
(3.2)
If the substrate
is positively charged, it follows from equation 3.1 that
(3.3)
(3.4)
where
(3.5)
Kmapp is the apparent Michaelis constant that
would be determined experimentally using the known bulk substrate
concentrations. If the substrate is negatively charged, the following
relationships hold:
(3.6)
(3.7)
The Km
of an enzyme for a substrate is apparently reduced if the substrate
concentration in the vicinity of the enzyme's active site is higher than that
measured in the bulk of the solution (Figure 3.7). This is because a lower bulk
concentration of the substrate is necessary in order to provide the higher
localised substrate concentration needed to half-saturate the enzyme with
substrate. Similar effects on the local concentration of products, inhibitors,
cofactors and activators may change the apparent kinetic constants involving
these molecules. For example, the apparent inhibition constant of a
positively-charged competitive inhibitor is given by,
(3.8)
Figure 3.7. The effect of immobilisation and
ionic strength on the Km of bromelain for its positively-charged
substrate, N-a-benzoyl-L-arginine ethyl ester. The support is the
negatively-charged poly-anionic polymer, carboxymethyl cellulose (Engasser &
Horvath, 1976). ——— free enzyme; ------ immobilised enzyme.
If the immobilised enzyme contains a number of groups
capable of chelating cations the partition of such cations into the
microenvironment is far greater than that described by the electrostatic
partition coefficient (j). For example, soluble glucose isomerase needs
a higher concentration of magnesium ions, than those required by the immobilised
enzyme. It also requires the presence of cobalt ions which do not need to be
added to processes involving the immobilised enzyme due to their strong
chelation by the immobilised matrix. A high concentration of ionising groups may
cause a partitioning of gases away from the microenvironment with consequent
effects on their apparent kinetic parameters. It is also a useful method for
protecting oxygen-labile enzymes by 'salting out' the oxygen from the vicinity
of the enzyme.
Differential partitioning of the components of redox couples may
have a significant effect on the activity and stability of certain enzymes. For
example, papain is stabilised by the presence of thiols, which act as effective
reducing agents. However, thiols possess partial negative charges at neutral pHs
which causes their expulsion from the microenvironment of papain if it is
immobilised on the negatively charged clay, kaolinite. In effect, the redox
couple involving thiol and uncharged disulphide, which is not partitioned,
becomes more oxidising around this immobilised enzyme. The net effect is a
destabilisation of the immobilised papain relative to the free enzyme.
pKa ≈ 8
2H+ + 2R-S−
2R-SH
R-S-S-R +
2H+ + 2e− [3.6]
reduced oxidised
(stabilised enzyme) (destabilised
enzyme)
Partition of hydrogen ions represents an
important case of solute partition. The pH of the microenvironment may differ
considerably from the pH of the bulk solution if hydrogen ions are partitioned
into or out of the immobilised enzyme matrix. The binding of substrate and the
activity of the immobilised enzyme both depend on the local microenvironmental
pH whereas the pH, as measured by a pH meter, always reflects the pH of the
bathing solution. This causes apparent shifts in the behaviour of the kinetic
constants with respect to the solution pH (Figure 3.8). It is quite a simple
process to alter the optimum pH of an immobilised enzyme by 1 - 2 pH units
giving important technological benefits (e.g., allowing operation of a process
away from the optimum pH of the soluble enzyme but at a pH more suited to the
solubility or stability of reactants or products).
Figure 3.8. Schematic diagram of the variation in the profiles
of activity of an enzyme, immobilised on charged supports, with the pH of the
solution.——— free enzyme. -------
enzyme bound to a positively charged
cationic support; a bulk pH of 5 is needed to produce a pH of 7 within the
microenvironment. ------- enzyme bound to a negatively charged anionic support;
a pH of 7 within the microenvironment is produced by a bulk pH of 9.
In addition to its affect on solute partition, the
localised electrostatic gradient may affect both the Km and
Vmax by encouraging or discouraging the intramolecular approach of
charged groups within the enzyme, or enzyme-substrate complex, during binding
and catalysis. A large number of small energetic gains and losses may complicate
the analysis of such overall effects (Table 3.4). As the resultant changes are
also reduced by increases in the ionic strength of the solution, these
electrostatic effects may be difficult to distinguish from the effects of
partition.
Table 3.4 The effect of
covalent attachment to a charged matrix on the kinetic constants of chymotrypsin
for N-acetyl-L-tyrosine ethyl ester (Goldstein
1972)
|
Ionic strength
(M)
|
kcat
(s−1)
|
Km
(mM
|
specificity
(kcat/Km)
|
Free enzyme
|
0.05
|
184
|
0.74
|
249
|
1.00
|
230
|
0.55
|
418
|
Enzyme attached to a
negatively charged support
|
0.05
|
300
|
2.50
|
120
|
1.00
|
280
|
1.93
|
145
|
Enzyme attached to a
positively charged support
|
0.05
|
119
|
7.10
|
17
|
1.00
|
1.65
|
5.82
|
28
|
The changes in kcat may be due to the approach of two positively
charged groups during the rate-controlling step in the catalysis
Hydrophobic interactions
play a central role in the structure of lipid membranes and the conformation of
macromolecules including enzymes. They are responsible for the relative
solubility of organic molecules in aqueous and organic solvents. These
interactions involve an ordered rearrangement of water molecules at the approach
of hydrophobic surfaces. The force of attraction between hydrophobic surfaces
decays exponentially with their distance apart, halving every nanometer
separation. These hydrophobic interactions effectively reduce the dielectric
constant of the microenvironment with consequent modification of the acidity
constants of acid and basic groups on the enzymes, substrates, products and
buffers (Figure 3.9). Similar effects may alter the acidity constants of key
substrate binding groups, so affecting the Km of the immobilised
enzyme for its substrates. Hydrophobic interactions are unaffected by the ionic
strength or pH of the solution but may be neutralised by the presence of
neighbouring hydrophilic groups where they are sufficient to dominate the
localised structure of the water molecules. Hydrophobic interactions may,
therefore, cause the partition of molecules between the bulk phase and the
microenvironment. If the surface of the immobilised enzyme particles is
predominantly hydrophobic, hydrophobic molecules will partition into the
microenvironment of the enzyme and hydrophilic molecules will be partitioned out
into the bathing solution. The reverse case holds if the biocatalytic surface is
hydrophilic. Partition causes changes in the local concentration of the
molecules which, in turn, affects the apparent kinetic constants of the enzyme
in a similar manner to that described for immobilised enzyme particles
possessing a net charge. An example of this effect is the reduction in the
Km of immobilised alcohol dehydrogenase for butanol. If the support is
polyacrylamide the Km is 0.1 mM but if the more hydrophobic copolymer
of methacrylate with acrylamide is used as the support, the Km is
reduced to 0.025 mM. In this example, no difference is noticed in the apparent
values for Km using ethanol, a more hydrophilic substrate. A similar
effect may be seen in the case of competitive inhibitors (Table
3.5). Gases
partitioned out from the microenvironment by the presence of a charged support (e.g., oxygen) are generally partitioned into the microenvironment by hydrophobic
supports
Figure 3.9. Schematic diagram showing the effect of a
hydrophobic support on the pH-activity profile of immobilised enzymes in
solutions of low ionic strength. ——— free enzyme;
-------enzyme
immobilised on a hydrophobic support. The effective decrease in the dielectric
constant for the microenvironment reduces the dissociation of charged groups,
increasing the pKa of carboxylic acids and reducing the pKa
of some basic groups.
Table 3.5 The
effect of immobilisation using a hydrophobic support on the relative competitive
inhibition of invertase
|
Invertase
|
Inhibition
constant (ki, mM)
|
Soluble
|
Bound to
polystyrene
(hydrophobic)
|
Aniline
(hydrophobic)
|
0.94
|
0.39
|
Tris-(hydroxymethyl)-aminomethane
(hydrophilic)
|
0.45
|
1.10
|
The Ki is reduced where both the inhibitor and
support are hydrophobic.
Other
specific partition effects are associated with particular immobilisation
supports. As examples: (a) The apparent Km of glucoamylase for maltose
is considerably reduced when the enzyme is immobilised on titanium activated
supports; such supports having a specific affinity for some poly-alcohols. (b)
Some polyphenolic resins have specific affinities for polysaccharides which
assist their partition into the microenvironment.
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This page was established in 2004 and last updated by Martin
Chaplin on
6 August, 2014
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