Download Chem*3560 Lecture 6: Allosteric regulation of enzymes

Chem*3560
Lecture 6: Allosteric regulation of enzymes
Metabolic pathways do not run on a continuous basis, but are regulated
according to need
Catabolic pathways run if there is demand for ATP; for example glycolysis may be
slowed down if beta oxidation is meeting current energy needs. Beta oxidation is more
suited for slow steady delivery of energy. Glycolysis supports need for rapid delivery of
energy.
Pathways for biosynthesis are also controlled
Is there a need for the product?
Is there sufficient energy or substrate or reducing power to carry out the
synthesis?
Where there are parallel catabolic and synthetic pathways, regulation is especially
important, because if both are allowed to run concurrently, it wastes energy:
Glycolysis: gain 2 ATP and 2 NADH
→
Glucose
2 pyruvate
←
Gluconeogenesis: cost 6 ATP + 2 NADH
If equivalent amounts of substrate are allowed to pass in each direction, 4 ATP would be
hydrolysed with no net benefit.
Glycolysis and gluconeogenesis are regulated in a reciprocal fashion so that each
pathway is only active if required, and then the opposite pathway is shut down.
Pathways are typically regulated near their starting point
Regulation near the start prevents substrate from being committed to the pathway when
product is not needed. If the pathway branches to yield two alternative products, then the
branch point is regulated so that only the product that is need is produced.
Typically an end product serves as the regulating ligand, a process known as negative
feedback.
1
2
3
4
A →! B →! C →!D → E
Reaction 1 is inhibited by product E
Enzymes may be regulated in several ways:
Allosteric regulation of substrate affinity;
Turn on or off by covalent modification of the enzyme;
Turn on by regulated expression of gene (transcription) or of synthesis of enzyme
(translation);
Turn off by regulated destruction of the enzyme (the amino acids are recycled and
used for other purposes).
The first two effects tend to give immediate responses to changing conditions. The last
two methods provide longer term responses.
The enzyme aspartate transcarbamoylase (ATCase) is a classic case of
allosteric regulation of affinity
ATCase catalyzes an early step in synthesis of pyrimidines
ATCase
more steps
carbamoyl phosphate + aspartate → carbamoyl aspartate → → uridine, cytidine
ATCase is negatively regulated by CTP
If CTP is present, there's no need to make more
This is an example of negative feedback control.
ATCase is positively regulated by ATP
If ATP is abundant, cells are growing, DNA and RNA synthesis are active
If ATP is abundant and [ATP] > [CTP], it's time to make more pyrimidines to
match the availability of purines.
ATCase shows sigmoidal kinetics with respect to substrate aspartate
Allosteric kinetics is described in terms of a low affinity T-state and high affinity R-state
When [Asp] is low, the enzyme is in T-state; as [Asp] increases, the enzyme switches to a
high affinity R-state. The enzyme is monitored in terms of the fraction of max rate of
catalysis, vo/Vmax, which is essentially a measure of the occupancy of the catalytic site.
With substrates only, the reaction rate
follows sigmoidal curve C.
When CTP binds (curve D), this
favours the T-state, so the curve is
shifted to the right (higher [Asp] is
needed to get the enzyme going).
When ATP binds (curve B), this
favours the R-state, so the curve is
shifted to the left (less [Asp] needed).
If [ATP] is high enough (curve A), there is no T-state, and ATCase follows the
hyperbolic curve of the pure R-state.
For allosteric enzymes, the substrate concentration giving vo/Vmax = 0.5 is designated as
K' or K0.5, and represents the apparent substrate affinity, just as P50 represents O2
affinity for hemoglobin. Although KM is derived in a similar manner, the term KM is not
used for allosteric enzymes because they don't follow the Michaelis-Menten equation.
T → R switch induced by
substrate occupancy is called
the positive homotropic effect
(the basic sigmoidal curve C
above).
Homotropic means literally
"change induced by the same
substance",
positive because affinity
increases with increasing [S],
ATP and CTP are allosteric
effectors for ATCase
T → R switch induced by binding
ATP is called a positive heterotropic
effect, and shifts the curve left..
Heterotropic means literally "change
induced by the another substance"
(other than a substrate).
R → T switch induced by
binding CTP is called a
negative heterotropic effect;
and shifts the curve right.
Negative because affinity
decreases with increasing
[CTP].
ATP and CTP are allosteric
effectors, substances that do
not participate directly in the
catalytic reaction, but which
modulate the allosteric behaviour of the enzyme to regulate the reaction.
When ATP and CTP are both present, they compete for the same binding site (see
below), and the relative concentration of the two nucleotides will determine whether a
positive or negative effect is observed. CTP has slightly more affinity, so [ATP] has to
be significantly higher than [CTP] for a positive effect.
Allosteric effectors are much more versatile than simple competitive or noncompetitive
inhibitors. When substrate concentration [S] is close to the enzyme's K0.5, a positive
effector can induce nearly full activity, while a negative effector can virtually turn the
enzyme off, exactly as desired for metabolic regulation.
ATCase is composed of two kinds of subunits
c (catalytic) subunits (33 kDa each) contain the substrate binding and catalytic site of the
enzyme, and can be in T or R catalytic states.
r (regulatory) subunits (17 kDa each) contain the ATP/CTP binding site and control the
activity of c subunits by determining their tendency to T or R state. r subunits have no
inherent catalytic activity.
The complete ATCase
molecule has formula c6r6
.
This can be broken down
into two modules of c3
(three catalytic sites) and
three modules of r2.
Each trimeric catalytic
module contains c subunits
placed every 120o around a
central axis (only one is
shown in the figure).
On its own, the c3 module
is catalytically active, but
unregulated, and with
normal Michaelis-Menten
hyperbolic kinetics.
The r2 module binds ATP
or CTP, but has no catalytic
activity of its own. The r2
modules bridge between
the c subunits of the upper
catalytic trimer and the
lower catalytic trimer .
The structure above is the
R state with substrates and
ATP bound . Below it is
the T state with sites
vacant.
In the T-state, the r2
modules reorient
themselves and bring the
catalytic units closer
together. This causes
interference between
projecting loops of the
catalytic subunits.