Chem*3560
Lecture 7: phosphofructokinase 1
Phosphofructokinase 1 is a key point for regulation of glycolysis
Glycogen → Glucose-1-P
↓
phosphofructokinase 1
Glucose → Glucose-6-P → Fructose-6-P → Fructose-1,6-bisP
↑
Fructose
→ etc
Phosphofructokinase 1 (PFK1) is the first enzyme that is common to the many possible starting
points for glycolysis, and is therefore a key point of regulation. PFK1 is a tetramer of four
simple subunits of 36 kDa (Lehninger p. 554), which can bind several allosteric effectors:
ATP is negative effector (promotes R → T switch), whose presence signals that cellular energy
supply is adequate. ATP is also substrate, and there are two binding sites for ATP in each
PFK1 subunit, one for substrate ATP adjacent to the fructose binding site, and one for
effector ATP quite distant from the substrate site. The effector ATP is not modified in
any way during the reaction at the catalytic site.
ADP (in bacteria) or 5'-AMP (in animals) is a positive effector (promotes T → R switch), whose
presence indicates that cellular energy may be low, and glycolysis is necessary to restore
ATP levels.
Citrate is negative effector whose presence indicates that the TCA cycle is well supplied with
its starting substrate, possibly derived from acetyl CoA coming from beta oxidation, and
hence glycolysis can be spared.
Phosphoenolpyruvate (PEP) is a negative effector which indicates that the end products of
glycolysis are accumulating, so no more substrate should be committed. High PEP may
also indicate that the metabolic balance is shifting in favour of gluconeogenesis, so
glycolysis enzymes such as PFK1 can be shut down.
Fructose-2,6-bisphosphate is a positive effector which can override other negative effectors.
Fructose-2,6-bisphosphate is a molecule made in small quantities by the enzyme
phosphofructokinase 2, and is otherwise not directly a part of any metabolic pathway
(Lehninger p.732). Synthesis of fructose-2,6-bisphosphate is a specific response to
hormones which signal that glycolysis should be turned on regardless of current energy
status in the cell.
Fructose2,6-bisphosphate plays an important role in coordinating the activities of
glycolysis versus gluconeogenesis, and will be dealt with in more detail later in the
semester.
5'-AMP is a sensitive indicator of cellular energy state:
Since ATP is normally present in cells at
about 1 mM, activity of PFK 1 is actually
more dependent on how well AMP or
fructose-2,6-bisphosphate can override ATP
induced inhibition.
The level of 5'-AMP is governed by the
equilibrium of the enzyme myokinase or
adenylate kinase.
ATP + AMP ! 2 ADP
K eq =
[ADP] 2
[ATP][AMP]
Keq = 2.2
rearranges to
[AMP] =
[ADP] 2
K eq [ATP]
Hence AMP concentration varies as the square of [ADP], and is thus a more sensitive indicator
of energy status.
Example: doubling the level of [ADP] causes [AMP] to increase more than fourfold.
If [ATP] = 1.0 mM, and [ADP] = 0.1 mM, then [AMP] = 0.12 / 2.2 = 0.0045 mM
If [ATP] = 0.90 mM, and [ADP] = 0.2 mM, then [AMP] = 0.04 / 2 = 0.02 mM
The response to fructose-2,6-bisphosphate is also dramatic
Fructose-2,6-bisphosphate, discovered in
1980, is the most powerful positive allosteric
effector for PFK1. 1 µM is sufficient to shift
the curve to the hyperbolic shape at extreme
left.
The second curve shows the response to ATP
as a substrate. The gray curve labelled MM
is what would be expected for ATP if it
behaved as a Michaelis-Menten substrate.
Because ATP binds not only to the catalytic
site as substrate, but also to the regulatory
site as a negative effector, the rate curve for
ATP drops off with increasing ATP.
Fructose-2,6-bisphosphate partly cancels the
drop off due to the negative effect of ATP.
Structure of Phosphofructokinase 1
PFK1 is a tetramer of four identical subunits (the R state is shown with substrate and effector
sites occupied by ADP).
Switch to T-state occurs by rotating the CD
subunit pair by about 10o relative to the AB
pair, so the switch is due a global change like that
of hemoglobin, and fits the MWC model of
allosteric behaviour.
Like hemoglobin, there is little change within the
AB subunit pair or within the CD pair.
The subunits of the tetramer show an interesting interdependence. One loop of subunit C
(green) is involved in binding the Fru-6-P into the catalytic site of subunit B (and similarly for all
the other substrate sites in subunits A, C and D. In addition, one loop of substrate D is involved
in binding ligand into the effector site of subunit C.
These interdependences provide a mechanism for communicating the allosteric state around the
four subunits of the tetramer.
The ligand in effector site C interacts with substrate in site B
The substrate site B and effector site C appear in the lower left and lower middle of the overall
image of PFK1 shown on the previous page. Similar interactions link substrate site A with
effector site B, substrate site C with effector site D, and substrate site D with effector site A
At the microscopic level, occupancy
of the effector site determines how the
loop from subunit C (green, right half
of figure) interacts with the substrate
site in B (cyan, left half of figure).
The presence of negative effector
causes the prominent helix in the upper
centre of the figure to unwind by one
turn. This lengthens the connecting
loop and arranges it so that Glu 161
faces the substrate site in the T-state.
Negative Glu repels negative
phosphate on Fru-6-phosphate, so
substrate affinity is lowered.
When a positive effector occupies the
site, the helix is extended by one turn
so that the loop is shorter, and now in
the R-state Arg 162 faces left into the
substrate site in the next subunit.
Positive Arg attracts negative
phosphate on Fru-6-phosphate, so
substrate affinity is increased.
If the effector sites are vacant, presence
of negative Fru-6-phosphate in the
substrate sites will tend to attract Arg
rather than Glu. If enough substrate
sites are occupied, the substrate can
induce the switch from T to R state (the
homotropic effect).
Supplementary image of ATCase (from lecture 6).
See Lehninger p. 279, which shows the T-state in space filling format)
The schematic version of ATCase shows the two c3 modules stacked together, but with access to
the substrate sites between the pair. The r2 modules form V shaped legs, and bridge between
pairs of c members from each of the c3 catalytic modules.
The transition between R and T has one global change, that is the two c3 modules are brought
closer together. The lower module rotates 15o relative to the upper module. When the c3
modules are close up in the T-state, access to substrate sites is partly obstructed, and peripheral
loops of the c structures interfere with each other in the gap. Hence affinity for substrate
decreases.
The transition is mediated through two effects on r2: the angle of the V closes up slightly,
and the orientation of the region of contact with c changes slightly so the Vs are at a slant
with respect to the central axis; this causes the rotation seen in the T-state.
T to R transition can be mediated by whether ATP or CTP occupies the effector site, in which
case the change is mediated by r2 modules and affects both c3 modules simultaneously..
T to R transition can also be mediated by substrate occupation of the catalytic sites, in which
case the change originates in the occupied c modules, which change their angle of attachment to
r, inducing the straighter r2 configuration of the R state. Because of the way the whole structure
is linked together, the transition must be all-or-none, so the effect is communicated to catalytic
sites that are still unoccupied.
Essentially the number of substrate sites occupied or effector sites occupied by ATP represent
votes in favour of R state. Presence of CTP or vacant substrate sites represent votes in favour of
T-state. The “votes” take the form of intramolecular forces that are induced by presence of
substrate or effector molecules.