Download Answers set 3

Chem*3560
1.
Question set 3
Jan 19-23, 2004
The three curves A, B, C
show the kinetic behaviour
of a typical allosteric enzyme
such as aspartate
transcarbamoylase
(ATCase).
Which of the curves
demonstrates a positive
homotropic effect?
Curves B and C are
sigmoidal, which is the
characteristic of the
homotropic effect.
Normal binding of a ligand to a site with constant affinity gives a hyperbolic curve. The
homotropic effect is the sigmoidal shape that arises because the protein is switching from
low affinity T state to high affinity R state due to the action of binding substrate itself.
This switch must occur while there are still vacant sites in a single molecule of protein, so only
proteins with multiple binding sites can show a homotropic effect. If a protein with a single
binding site undergoes induced fit changes, this can't affect the binding curve, because if the
single site is unoccupied, there's no effect, and if the site is occupied, there's no vacant site in the
same molecule to demonstrate that a change has taken place.
Which demonstrates a positive heterotropic effect?
A positive heterotropic effect is a shift to higher affinity due to binding a ligand other than
substrate that causes the protein to switch in favour of the R state. Increased affinity is
indicated by a shift to the left (less [S] needed to observe same activity). In the extreme case,
the ligand could convert the protein 100% to R state, so homotropic effect is no longer
observed. Curve A shows this effect.
Which demonstrates a negative heterotropic effect?
A negative heterotropic effect is a shift to lower affinity due to binding a ligand other than
substrate that causes the protein to switch in favour of the T state, or to stay in T state until
much higher [S] is present. Decreased affinity is indicated by a shift to the right (more [S]
needed to observe same activity). Curve C shows this effect.
A sigmoidal curve is the sign of a homotropic effect.
Effectors that shift the curve to left or right indicate a heterotropic effect.
Chem*3560
2.
Question set 3
Jan 19-23, 2004
How was the quarternary structure of aspartate transcarbamoylase discovered?
The exact details were found by X-ray crystallography, the standard method for determining
molecular structure of protein (Lehninger p.279). However the first clue was obtained by
treatment with mercury compounds that react with Cys -SH groups. These abolish the
allosteric behaviour, because they break up the complete c6 r6 molecule into the c3 and r2
modules (Stryer p. 239).
3.
What is the logic behind the finding that ATP is a negative effector of aspartate
transcarbamoylase, while CTP is a positive effector?
Very bad logic! ATP is a positive effector and CTP is a negative effector.
Aspartate transcarbamoylase is the first step on the pathway that makes pyrimidine nucleosides,
including cytidine and uridine. Pyrimidines are needed for RNA and DNA synthesis. Presence
of CTP, cytidine triphosphate, indicates that enough cytidine is already availble, so there is no
need to invest substrate and energy in making more.
Presence of ATP in the effector site means that the relative concentration of ATP is higher than
CTP. DNA and RNA synthesis are likely to need similar quantities of purine and pyrimidine
nucleotides, so some more pyrimidine should now be made to bring purines and pyrimidines
back into balance. Also presence of high ATP indicates that the energy status of the cell is
good, leading to growth and a need for nucleotides for DNA and RNA synthesis.
4.
The graph on the right show how reaction rate of
phosphofructokinase 1 varies as a function of its
second substrate, ATP.
Fructose-6-P + ATP →Fructose-1,6-bisP + ADP
Why does reaction rate decrease at high [ATP]?
ATP is a substrate of the enzyme, so one molecule of ATP must bind in the catalytic site.
The broken line (typical Michaelis-Menten hypeerbolic curve) shows the binding pattern for the
substrate site, and how [ATP] would determine catalytic activity if allosteric effects could be
ignored. The curve is hyperbolic because there is no homotropic effect due to ATP in the
substrate site (this means that ATP in the substrate site has no influence on the balance between
T and R states).
However, PFK1 has a second binding site for ATP which is an allosteric effector site, and ATP
is a negative effector. As [ATP] increases, ATP starts to fill the effector site and switch the
enzyme to a less active T state. As a result activity actually goes down with increasing [ATP].
Chem*3560
5.
Question set 3
Fill out the table with yes or no as appropriate
ATCase subunit
c6r6
c3
Shows catalytic activity
Allosteric behaviour
yes
yes
yes
no
Binds CTP or ATP
yes
Binds substrates
yes
ATP as
substrate
yes
Jan 19-23, 2004
r2
c
r
no
r2 has a shape change
but no catalytic
activity to detect the
consequence of shape
change
yes
no
no
no
no
no
no
no
no
no
The substrate sites lie at the boundary between subunits in c3 , so that part of eac site is
contributed by the neighbouring subunit. In a single c subunit, the substrate binding site is
incomplete.
6.
Distinguish between the terms R-subunit and R-state of an allosteric enzyme.
One of the subunits of protein kinase A is called the R subunit, and this is common nomenclature
for enzymes with a distinct molecular component that plays a regulatory role rather than a
catalytic role. In the case of protein kinase A, when R subunit binds to the catalytic C subunit, it
masks the active site so that the enzyme appears to lack catalytic activity. In the case of cyclin
dependent kinase, cyclin could be considered to be the R subunit, but one that activates when it
binds, by helping arrange the catalytic site in a correct configuration.
R-state refers to an alternative conformation or arrangement of the polypeptide containing the
catalytic site in an enzyme showing allosteric cooperative behaviour such as ATCase or PFK1.
R-state usually refers to the high affinity or high activity state, while T-state referes to the low
affinity or low activity state.
The terminology R and T states is derived from the structural events in hemoglobin, in which the
low affinity state exists in a state of tension (T for Tense) between the O2 -binding Fe atom and
Helix F. The R state is Relaxed, and allows the Fe atom to adopt the best position for binding
O2
Chem*3560
7.
Question set 3
Jan 19-23, 2004
Phosphonoacetyl-L-aspartate closely resembles the transition state for the the reaction
of carbamoyl phosphate and aspartate. Why does it act as a competitive inhibitor of
ATCase at high substrate concentration, but stimulates activity at very low substrate
concentration?
Competitive inhibitors are typically molecules that are “look-alike” for the substrate, but don’t
allow the intended reaction. Therefore they occupy the substrate binding site, keeping real
substrate out. Phosphono-L-aspartate is a sufficiently similar look alike that it binds to the same
amino acids as real substrate, and can induce T to R switch.
ATCase has six substrate binding sites per c6 r6 enzyme. When substrate concentration is very
low so that the mean occupancy in ATCase is one or two sites, the enzyme stays in the T or low
activity state. Phosphono-L-aspartate can occupy some of the other sites in the c6 r6 enzyme,
and help induce switch to high activity R state. So although occupancy of some catalytic sites
by phosphono-L-aspartate is excluding substrate from those sites, the switch to R-state is more
than compensating for the inhibition, and the overall effect is to stimulate activity.
At high substrate concentration, the substrate would be expected to occupy all sites, so the
effect of phosphono-L-aspartate in excluding substrate does result in inhibition as expected.