BCH 401 – ADVANCED ENZYMOLOGY

Allosteric enzymes
An allosteric protein is one in which the binding of a ligand to one site affects the
binding properties of another site on the same protein.
- Allosteric enzymes do not obey Michaelis-Menten kinetics.
- A plot of V0 against [S] for an allosteric enzyme gives a sigmoidal curve rather than
the hyperbolic plots predicted by the Michaelis–Menten equation. The curve has a steep
section in the middle of the substrate concentration range, reflecting the rapid increase
in enzyme velocity which occurs over a narrow range of substrate concentrations. This
allows allosteric enzymes to be particularly sensitive to small changes in substrate
concentration within the physiological range.
- In allosteric enzymes, the binding of a substrate molecule to one active site affects the
binding of substrate molecules to other active sites in the enzyme; the different active
sites are said to behave cooperatively in binding and acting on substrate molecules,
meaning that the binding reactions at individual sites are not independent of one
another. Thus allosteric enzymes are often multisubunit proteins, with one or more
active sites on each subunit. The allosteric enzyme has an oligomeric quaternary
structure (i.e. dimer, trimer, tetramer etc.). The binding of substrate at one active site
induces a conformational change in the protein that is conveyed to the other active
sites, altering their affinity for substrate molecules.

In allosteric enzymes, the binding of substrate to one active site can alter the properties
of other active sites in the same enzyme molecule. A possible outcome of this interaction
between subunits is that the binding of substrate becomes cooperative; the binding of
substrate to one active site facilitates the binding of substrate to the other active sites
resulting in the sigmoidal plot of V 0 versus substrate concentration. The activity of an
allosteric enzyme may be altered by regulatory molecules that are reversibly bound to
specific site other than the catalytic sites. The catalytic properties of allosteric enzymes
can thus be adjusted to meet the immediate needs of a cell. For this reason, allosteric
enzymes are key regulators of metabolic pathways.

- Positive cooperativity confers the metabolic advantage of amplifying the sensitivity of

a signal, i.e. a small change in ligand L can have far greater effect on the output
response in a positive cooperativity system than in Michaelis-Menten system. For
example, a three-fold increase in the ligand (L = O 2) concentration for haemoglobin
changes the binding capacity nine-fold. For Michaelis-Menten this would require 81-
fold change in ligand concentration.

Myoglobin and haemoglobin: classic example of allostery

- Myoglobin and haemoglobin are oxygen transport and storage proteins, respectively.
- Myoglobin is monomeric, whereas haemoglobin is tetrameric
- For myoglobin, a binding curve indicating a simple chemical equilibrium is observed.
The curve rises sharply as pO2 increases and then levels off. Half-saturation (P50) is at 2
toor (mm Hg) which is very low and indicates that oxygen binds with high affinity to
myoglobin
- For haemoglobin, the binding curve resembles a sigmoid shape. Half-saturation is at
26 torr which indicates that haemoglobin has a low oxygen affinity. This is due to a
haemoglobin interaction with a molecule called 2,3-bisphosphoglycerate (2,3-BPG),
which significantly lowers haemoglobin oxygen affinity.
- The physiological significance of cooperative binding of oxygen by hemoglobin:
o Oxygen must be transported in the blood from the lungs
o Oxygen must be released in the capillaries
o If hemoglobin behaved like myoglobin, very little oxygen would be released in
capillaries
o In the lungs, hemoglobin becomes nearly saturated with oxygen such that 98% of the
oxygen-binding sites are occupied. When hemoglobin moves to the tissues and releases
O2, the saturation level drops to 32%. Thus, a total of 66% of the potential oxygen-
binding sites contribute to oxygen transport. The cooperative release of oxygen favors
unloading of oxygen in tissues. If myoglobin were to transport oxygen, it would be 98%
saturated in the lungs but would remain 91% saturated in the lungs, only 7% of the
sites would contribute to oxygen transport.
o The sigmoid, cooperative oxygen binding curve of hemoglobin makes this possible

MWC and KNF models of allosterism

- Two limiting models have been developed to explain the cooperative binding of ligands
to a multi-subunit assembly such as hemoglobin.
Two models for the cooperative binding of ligands to proteins with multiple binding sites
have greatly influenced thinking about this problem.

- MWC model – concerted model (Monod, Wyman, Changeux 1965)

The symmetry or concerted model was first proposed by Jacques Monod, Jeffries
Wyman and Jean-Pierre Changeaux in 1965 (sometimes referred to as the Monod-
Wyman-Changeaux (MWC) model). The concerted model assumes that the subunits of a
cooperatively binding protein are functionally identical, that the subunits of an allosteric
enzyme can exist in one of only two states, T and R. T-state subunits are in a tense
state that is compact and relatively inactive, while R- state subunits are in a relaxed,
expanded, active state with higher affinity for the substrate; no intermediate states are
allowed. In the absence of bound substrate, the equilibrium favors the T-state. As
substrate binds to each active site in the T-state, the equilibrium shifts towards the R-
state. All of the subunits change conformation in a concerted manner, which implies
that the conformation of each subunit is constrained by its association with the other
subunits; in other words, there are no oligomers that simultaneously contain R- and T-
state subunits and the molecular symmetry of the protein is conserved during the
conformational change.

Models of allosterism. (a) The concerted or symmetry model; the squares and circles represent the T- and R- states,
respectively. (b) The sequential model; substrate binding progressively induces changes in the subunits.

- KNF model – sequential model (Koshland, Nemethy, Filmer 1966)

In the alternative sequential model (KNF model), first proposed by Daniel Koshland and
his colleagues Nemethy and Filmer (1966), sequential changes in structure take place
within an oligomeric enzyme as the individual active sites are occupied. The binding of
substrate to one site influences the substrate affinity of neighboring active sites without
necessarily inducing a transition encompassing the whole enzyme, such that the
molecular symmetry of the whole protein is not necessarily conserved ( Fig. b). The
sequential model builds upon the induced-fit hypothesis of enzyme–substrate
interaction, whereas the concerted model implicitly assumes the lock-and-key model of
substrate binding to the enzyme’s active site. In the sequential model, substrate binding
induces a conformational change in a subunit and cooperative interactions arise through
the influence that these conformational changes have on neighboring subunits. The
strengths of these interactions depend on the degree of mechanical coupling between
subunits. In the sequential model the enzyme– substrate binding affinity varies with the
number of bound substrate molecules, whereas in the concerted model this affinity
depends only on the quaternary state of the enzyme. The results of studies of a number
of allosteric proteins suggest that most behave according to a combination of the
concerted and sequential models i.e. the two models are not mutually exclusive.

o Glyceraldehyde-3-phsphate dehydrogenase is a homotetramer that shows negative

+
cooperativity in binding the homotropic ligand NAD such that binding of the fourth
+
NAD is decreased by 300,000 compared to that of the first.
o MWC cannot explain this since if the symmetry constraint applies, binding of the
+ +
fourth NAD will convert all four subunits to the T state and consequently all the NAD
will be released again since it is now 300,000 times less firmly bound
o The KNF model is more general and can explain positive and negative cooperativity.
The MWC is thus a simplified form of the KNF model
o MWC explains the behavior of 2,3-BPG on hemoglobin more elegantly than does KNF.
However, the KNF model is closer approximation to the real situation and will also
describe negative cooperativity and the behavior of heteromeric enzymes.

Allosteric enzymes may be controlled by effector molecules (activators and inhibitors)

that bind to the enzyme at a site other than the active site (either on the same subunit
or on a different subunit), thereby causing a change in the conformation of the active
site which alters the rate of enzyme activity. An allosteric activator increases the rate of
enzyme activity, while an allosteric inhibitor decreases the activity of the enzyme.

Hemoglobin binding regulation

- In the absence of 2,3-BPG, oxygen binding to hemoglobin follows a rectangular
hyperbolar (O2 binds much tighter and doesn’t release easily)
- 2,3-BPG binds deoxyhemoglobin and acts to stabilize the low oxygen affinity state (T
state)
- Since 2,3-BPG binds at a site distant from the Fe where oxygen binds, it is called an
allosteric effector
- 2,3-BPG is highly negatively charged and stabilized the T-state with salt bridges
- 2,3-BPG is synthesized in erythrocytes and reduces O 2 affinity of hemoglobin thus
helps the adaptation of humans to environments with different pO 2 (sea level versus
high altitude). Pure hemoglobin binds oxygen more tightly than does hemoglobin in red
blood cells this difference is due to the presence of 2,3-BPG in red blood cells.
- 2,3-BPG binds in the center of the tetramer in the central activity.
- Fetal hemoglobin has a lower affinity for 2,3-BPG than maternal hemoglobin (=
higher affinity for oxygen), so oxygen can pass from mother to fetus.

Bohr Effect
- Under normal conditions, when tissues are metabolically active there is a high level of
CO2 and a low pH. When tissues are metabolically inactive, there is a low level of CO 2
and a high pH.
- Rapidly metabolizing tissues, such as contracting muscle, generate large amounts of
hydrogen ions and CO2. To release oxygen where the need is greatest, hemoglobin has
evolved to respond to higher levels of these substances. Hydrogen ions and CO 2 are
allosteric effectors of hemoglobin that bind to sites on the molecule that are distinct
from the oxygen-binding sites.
- The regulation of oxygen binding by hydrogen ions and CO2 is called the Bohr effect
after Christian Bohr, who described it in 1904
+
- Hemoglobin caries H and CO2 (end products of respiration) back to the lungs. The
binding of protons diminishes oxygen binding by favoring the T state through
protonation of a histidine residue. And the binding of oxygen diminishes proton binding.
- In the second mechanism, the hydration of CO 2 in tissues and extremities leads to
proton production. CO2 decreases the affinity of hemoglobin for oxygen even beyond the
effect due to a decrease in pH, resulting in even more efficient oxygen transport from
the tissues to the lungs. These protons are taken up by hemoglobin as oxygen dissociates.
The reverse occurs in the lungs.
- CO2 also binds to hemoglobin directly at the amino-terminal end of each globin chain
and stabilizes the t state via additional salt bridges
- Now looking at the Bohr effects on metabolic activity

o When tissues are metabolically active, hemoglobin gives up O 2 causing a high CO2/O2
exchange
o When tissues are metabolically inactive, hemoglobin holds unto O 2 causing a low
CO2/O2 exchange
Other allosteric proteins
- PFK-1 is negatively regulated by ATP and citrate and positively by ADP/AMP and
fructose-2,6-bisphosphate
- Protein kinase A, cAMP binding exhibits positive cooperativity
- Glycogen phosphorylase is inhibited by ATP and glucose-6-phosphate. Phosphate is a
positive homotropic effector (if stimulates binding of itself
- Pyruvate dehydrogenase is inhibited by ATP and CoA and NADH (end products of the
+
citric acid cycle) and activated by AMP, CoA and NAD
 Enzyme Assays and Enzyme Purification

Enzyme Assays
Enzyme assays are laboratory methods for measuring enzymatic activity. Enzyme
activity is a measure of the ability of a given enzyme to convert its substrate(s) into its
product(s). Depending on the enzyme it is typically assayed by measuring either the
amount of substrate that is disappearing, or the amount of product that is appearing
over a specified period of time, such that the final result is expressed in terms of moles
of conversion per unit mass of protein per unit time, for example, µmole of product per
minute or nmol/mg protein/sec.
Under the usual in vitro assay conditions, the enzyme is present in limiting or
M while [S] is generally 10
-12 -7 -6
“catalytic” amounts in the neighborhood of 10 to 10
M. The initial velocity (Vi) is always directly proportional to total enzyme [E]t,
-2
to 10
and this fact can be used to quantitate the amount of enzyme present. It should be
stressed that the relationship between Vi and [E]t is linear only if true initial velocities
are measured. Since Vi varies with [S], the assay period must be short enough to ensure
that only a small fraction of the substrate is utilized (5% or less).

Types of Assay
All enzyme assays measure either the consumption of substrate or production of
product over time. A large number of different methods of measuring the
concentrations of substrates and products exist and many enzymes can be assayed in
several different ways. Biochemists usually study enzyme-catalyzed reactions using four
types of experiments:

Initial Rate Experiments

When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate
intermediate builds up in a fast initial transient. Then the reaction achieves a steady-
state kinetics in which enzyme substrate intermediates remains approximately constant
over time and the reaction rate changes relatively slowly. Rates are measured for a
short period after the attainment of the quasi-steady state, typically by monitoring the
accumulation of product with time. Because the measurements are carried out for a
very short period and because of the large excess of substrate, the approximation free
substrate is approximately equal to the initial substrate can be made. The initial rate
experiment is the simplest to perform and analyze, being relatively free from
complications such as back-reaction and enzyme degradation. It is therefore by far the
most commonly used type of experiment in enzyme kinetics.

Progress Curve Experiments

In these experiments, the kinetic parameters are determined from expressions for the
species concentrations as a function of time. The concentration of the substrate or
product is recorded in time after the initial fast transient and for a sufficiently long
period to allow the reaction to approach equilibrium. We note in passing that, while
they are less common now, progress curve experiments were widely used in the early
period of enzyme kinetics.

Transient Kinetics Experiments

In these experiments, reaction behavior is tracked during the initial fast transient as the
intermediate reaches the steady-state kinetics period. These experiments are more
difficult to perform than either of the above two classes because they require rapid
mixing and observation techniques.

Relaxation Experiments
In these experiments, an equilibrium mixture of enzyme, substrate and product is
perturbed, for instance by a temperature, pressure or pH jump, and the return to
equilibrium is monitored. The analysis of these experiments requires consideration of the
fully reversible reaction. Moreover, relaxation experiments are relatively insensitive to
mechanistic details and are thus not typically used for mechanism identification,
although they can be under appropriate conditions.

Continuous vs. Discontinuous Assays

Enzyme assays can be split into two groups according to their sampling method:
continuous assays, where the assay gives a continuous reading of activity, and
discontinuous assays, where samples are taken, the reaction stopped and then the
concentration of substrates/products determined.

Continuous Assays
Continuous assays are most convenient, with one assay giving the rate of reaction with
no further work necessary. There are many different types of continuous assays.
1. Spectrophotometric
In spectrophotometric assays, you follow the course of the reaction by measuring a
change in how much light the assay solution absorbs. If this light is in the visible region
you can actually see a change in the color of the assay, these are called colorimetric
assays. The MTT assay, a redox assay using a tetrazolium dye [MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble purple
formazan, (E,Z)-5-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) as
substrate is an example of a colorimetric assay.
Also, UV light is often used since the common coenzymes NADH and NADPH absorb UV
light in their reduced forms, but do not in their oxidized forms. An oxidoreductase using
NADH as a substrate could therefore be assayed by following the decrease in UV
absorbance at a wavelength of 340 nm as it consumes the coenzyme.

2. Fluorometric
Fluorescence is when a molecule emits light of one wavelength after absorbing light of a
different wavelength. Fluorometric assays use a difference in the fluorescence of
substrate from product to measure the enzyme reaction. These assays are in general
much more sensitive than spectrophotometric assays, but can suffer from interference
caused by impurities and the instability of many fluorescent compounds when exposed
to light.
An example of these assays is again the use of the nucleotide coenzymes NADH and
NADPH. Here, the reduced forms are fluorescent and the oxidized forms non-
fluorescent. Oxidation reactions can therefore be followed by a decrease in fluorescence
and reduction reactions by an increase. Synthetic substrates that release a fluorescent
dye in an enzyme-catalyzed reaction are also available, such as 4-methylumbelliferyl-
β-D-galactoside for assaying β-galactosidase.

3. Calorimetric
Calorimetry is the measurement of the heat released or absorbed by chemical reactions.
These assays are very general, since many reactions involve some change in heat and
with use of a microcalorimeter, not much enzyme or substrate is required. These assays
can be used to measure reactions that are impossible to assay in any other way.

4. Chemiluminescent
Chemiluminescence is the emission of light by a chemical reaction. Some enzyme
reactions produce light and this can be measured to detect product formation. These
types of assay can be extremely sensitive, since the light produced can be captured by
photographic film over days or weeks, but can be hard to quantify, because not all the
light released by a reaction will be detected. The detection of horseradish peroxidase by
enzymatic chemiluminescence (ECL) is a common method of detecting antibodies in
western blotting. Another example is the enzyme luciferase, this is found in fireflies and
naturally produces light from its substrate luciferin.

5. Light Scattering
Static light scattering measures the product of weight-averaged molar mass and
concentration of macromolecules in solution. Given a fixed total concentration of one or
more species over the measurement time, the scattering signal is a direct measure of
the weight-averaged molar mass of the solution, which will vary as complexes form or
dissociate. Hence the measurement quantifies the stoichiometry of the complexes as well
as kinetics. Light scattering assays of protein kinetics is a very general technique that
does not require an enzyme.

6. Microscale Thermophoresis
Microscale thermophoresis (MST) measures the size, charge and hydration entropy of
molecules/substrates in real time. The thermophoretic movement of a fluorescently
labeled substrate changes significantly as it is modified by an enzyme. This enzymatic
activity can be measured with high time resolution in real time. The material
consumption of the all optical MST method is very low, only 5 μl sample volume and
10 nM enzyme concentration are needed to measure the enzymatic rate constants for
activity and inhibition. MST allows the measurement of the modification of two
different substrates at once (multiplexing) if both substrates are labeled with different
fluorophores. Thus substrate competition experiments can be performed.

Discontinuous Assays
Discontinuous assays are when samples are taken from an enzyme reaction at intervals
and the amount of product production or substrate consumption is measured in these
samples.
1. Radiometric
Radiometric assays measure the incorporation of radioactivity into substrates or its
release from substrates. The radioactive isotopes most frequently used in these assays are
14C, 32P, 35S and 125I. Since radioactive isotopes can allow the specific labelling of a
single atom of a substrate, these assays are both extremely sensitive and specific. They
are frequently used in biochemistry and are often the only way of measuring a specific
reaction in crude extracts (the complex mixtures of enzymes produced when you lyse
cells). Radioactivity is usually measured in these procedures using a scintillation counter.

2. Chromatographic
Chromatographic assays measure product formation by separating the reaction mixture
into its components by chromatography. This is usually done by high-performance liquid
chromatography (HPLC), but can also use the simpler technique of thin layer
chromatography. Although this approach can need a lot of material, its sensitivity can
be increased by labelling the substrates/products with a radioactive or fluorescent tag.
Assay sensitivity has also been increased by switching protocols to improved
chromatographic instruments (e.g., ultra-high pressure liquid chromatography) that
operate at pump pressure a few-fold higher than HPLC instruments.

Factors to Control in Enzyme Assays

1 Salt Concentration
Most enzymes cannot tolerate extremely high salt concentrations. The ions interfere
with the weak ionic bonds of proteins. Typical enzymes are active in salt concentrations
of 1-500 mM. As usual there are exceptions such as the halophilic (salt loving) algae
and bacteria.

2 Effects of Temperature
All enzymes work within a range of temperature specific to the organism. Increases in
temperature generally lead to increases in reaction rates. There is a limit to the increase
because higher temperatures lead to a sharp decrease in reaction rates. This is due to
the denaturating (alteration) of protein structure resulting from the breakdown of the
weak ionic and hydrogen bonding that stabilize the three dimensional structure of the
enzyme active site. The "optimum" temperature for human enzymes is usually between
35 and 40°C. The average temperature for humans is 37°C. Human enzymes start to
denature quickly at temperatures above 40°C. Enzymes from thermophilic archaea
found in the hot springs are stable up to 100°C. However, the idea of an "optimum"
rate of an enzyme reaction is misleading, as the rate observed at any temperature is
the product of two rates, the reaction rate and the denaturation rate. If you were to
use an assay measuring activity for one second, it would give high activity at high
temperatures, however if you were to use an assay measuring product formation over
an hour, it would give you low activity at these temperatures.

3 Effects of pH
Most enzymes are sensitive to pH and have specific ranges of activity. All have an
optimum pH. The pH can stop enzyme activity by denaturating (altering) the three
dimensional shape of the enzyme by breaking ionic, and hydrogen bonds. Most enzymes
function between a pH of 6 and 8; however pepsin in the stomach works best at a pH
of 2 and trypsin at a pH of 8.

4 Substrate Saturation
Increasing the substrate concentration increases the rate of reaction (enzyme activity).
However, enzyme saturation limits reaction rates. An enzyme is saturated when the
active sites of all the molecules are occupied most of the time. At the saturation point,
the reaction will not speed up, no matter how much additional substrate is added. The
graph of the reaction rate will plateau.

5 Macromolecular Crowding
The phenomenon of macromolecular crowding alters the properties of molecules in a
solution when high concentrations of macromolecules such as proteins are present. Such
conditions occur routinely in living cells; for instance, the cytosol of Escherichia coli
contains about 300–400 mg/ml of macromolecules. Crowding occurs since these high
concentrations of macromolecules reduce the volume of solvent available for other
molecules in the solution, which has the result of increasing their effective
concentrations.
This crowding effect can make molecules in cells behave in radically different ways than
in test-tube assays. Consequently, measurements of the properties of enzymes or
processes in metabolism that are made in the laboratory ( in vitro) in dilute solutions
may be different by many orders of magnitude from the true values seen in living cells
(in vivo). The study of biochemical processes under realistically crowded conditions is
very important, since these conditions are a ubiquitous property of all cells and
crowding may be essential for the efficient operation of metabolism.

General Purification Scheme

Enzymes are purified by employing successive chemical or physical fractionation
procedures. The object of each step is to retain as much of the desired enzyme as
possible while getting rid of as much of the other proteins, nucleic acids, and the like, as
possible. The efficiency of each step is given by the “yield” or “recovery” (the percent of
the total enzyme activity originally present that is retained) and the ‘purification” or
“purification factor” (the factor by which the specific activity of the preparation has
increased). The object is to optimize both factors. Sometimes a good yield is sacrificed
for the sake of an excellent purification step; sometimes a good purification step is not
used because the yield is too low.
A hypothetical purification scheme is shown in Table below. The crude cell-free extract
may be prepared by a number of means depending on the nature of the starting tissue
or cells and the size of the preparation. Some common cell-breakage methods include (i)
autolysis, (ii) freeze-thaw, (iii) sonic, (iv) oscillation, (v) mechanical grinding, (with or
without an abrasive), (vi) ballistic homogenization, or (vii) disruption in any one of a
number of pressure cells (Xpress, French press). The resulting homogenate is usually
centrifuged to remove unbroken cells and large debris.
There are no general rules concerning the order of the purification steps although heat
treatment (where possible) and ammonium sulfate precipitation are usually done early
in the purification sequence. Gel filtration can follow ammonium sulfate precipitation
and, thereby, serve to desalt the preparation as well as fractionate the proteins
according to size. If ion-exchange chromatography is to follow the ammonium sulfate
step, then it is a good idea to dialyze the preparation first, or pass the preparation
through a rapid gel filtration column (e.g., Sephadex G-25). The removal of the
ammonium sulfate will facilitate the binding of the proteins to the ion-exchange
column. Other steps not shown in the purification table that may be highly effective for
certain enzymes include (i) differential centrifugation (for mitochondria, chloroplasts,
nuclei, mitosomes, ribosomes), (ii) pH precipitation, (iii) organic solvent precipitation
(e.g., ethanol, acetone), (iv) protamine sulfate or streptomycin sulfate precipitation (to
precipitate nucleic acids and acidic proteins), (v) affinity chromatography, and (vi)
preparative gel electrophoresis.

Table: Hypothetical enzyme purification scheme

The purity of the final preparation should be checked by several methods before
concluding that the preparation is homogenous. Suitable methods include (i) analytical
disc gel electrophoresis at several pH values and gel concentrations, and (ii)
ultracentrifugation. A homogenous enzyme preparation should elute from an ion-
exchange or gel filtration column as a single symmetrical activity and protein peak with
a constant specific activity throughout. A homogeneous preparation is by no means
necessary for kinetic analyses, but the purer the enzyme, the less the complications
from the competing reactions that may use up the substrate or the product.