Lecture 7: Regulation, Enzymes and Rate Limiting Steps

Learning Outcomes:

  • Describe the chemical features of ATP which make it ideal for use as an energy currency

  • Explain the concept of energy charge with reference to the concentration of adenine nucleotides

  • Review how a small change in ATP concentration is translated into a large relative change in AMP concentration

  • Identify the most likely control points in metabolic pathways

  • Interpret enzyme kinetic parameters to identify potential rate limiting steps

  • Describe the properties of rate limiting steps

  • Review the major ways in which enzyme activity can be changed

  • List the key rate limiting steps in the major pathways of catabolism

  • Provide an overview of the regulation of phosphofructokinase

  • Provide an overview of the regulation of hexokinase

  • Using an example, illustrate how control motifs act synergistically to regulate pathways

  • Using an example, show how enzymes are controlled by reversible phosphorylation

  • Recognise that rate limiting steps can change with circumstances

  • Explain the principles of reciprocal regulation of pathways

  • Cells are always checking how much energy they have – need to keep ATP at 5mM

    • Large changes in ATP are not desirable

  • ATP is not the most high energy molecule in our cells:

  • Higher energy molecules can transfer their phosphate group onto ADP – substrate level phosphorylation

    • Creatine phosphate – when you want to start sprinting quickly – burst of ATP that wasn’t stored in cells

      • Passes phosphate onto ADP as it is in a higher energy state

    • 1,3-Bisphosphoglycerate (second half of glycolysis – energy return phase)

      • 3 carbon sugar phosphate and adding another phosphate to it

    • Phosphenolpyruvate (PEP)

      • Also at the end of glycolysis

      • Very high energy phosphate bond

    • Above 3 molecules are instant reserves of high energy phosphate – can only supply for a few seconds

  • ATP:

    • energy released when any terminal phosphates are hydrolysed

      • ATP to ADP releases energy

      • ATP to AMP releases energy

  • Instant ATP:

    • Adenylate kinase (enzyme)

      • Take 2ADP and create an ATP and AMP

  • Energy Charge = measure of how much energy is in a cell – ratio of adenine nucleotide concentrations

  • AMP only appears on the bottom so large changes to AMP will reduce the energy charge of a cell significantly

  • Concentration of ATP in cells depends on type of cell and the organism – not always 5mM

    • BUT [ATP] > [ADP] > > [AMP]

    • AMP concentration is always very low

  • Energy Charge and AMP:

    • AMP is the most sensitive watch on energy charge

    • Has the largest relative change when all energy is depleted in cells

    • Easy for cells to watch AMP to signal for energy charge getting low

Which enzymes are controlled:

  • Slowest enzyme in the pathway determines the overall speed

    • Rate-limiting steps

    • Flux generating steps

      • Flux = flow of molecules on a pathway

    • Don’t need to control every enzyme involved

  • These enzymes being controlled are usually low Km

    • Don’t have a high reaction velocity

    • Usually working at substrate concentrations that are much higher than their Km value

    • At high substrate concentrations, minor changes in substrate concentrations will not affect the rate of the reaction

    • Doubling or halving the substrate concentration isn’t even going to effect the rate

    • The only time substrate concentration concentration impacts reaction rate is when it reaches Km value

    • Km = substrate concentration at which the enzyme is catalysing the reaction at half the maximum reaction velocity

    • When you are near Km, small changes in substrate concentration is going to effect the rate of reaction

    • x10 or x50 Km – changing substrate concentration will not effect the reaction velocity

Properties of RLS (rate limiting steps):

  • Irreversible

    • Need alternative enzymes to go back

    • Not equilibrium – not effected by substrate or product concentrations

    • Committed steps – once you’ve gone past a point, theres no way to get back without another enzyme

  • Saturated with substrate

    • Low kM or [substrate] > Km

    • Working at Vmax

  • e.g. Peak hour at train station barriers – barrier = enzyme working at maximum velocity – lots of people (substrate)

    • Increasing or decreasing passengers isn’t going to change the rate at which people go through the gate

    • Controlling one gate (enzyme) impacts how long all the other people take to get home or to their work (substrate)

3 major ways to regulate RLS:

  1. Change the intrinsic activity of the step

    1. Make the rate limiting enzyme go faster or slower

    2. Make ticket reading/gate opening happen faster

  2. Make more gates open

    1. Turn the rate limiting enzyme on/off or make it work the other way

    2. Switch gates from off to on

    3. Or change the direction from in to out

  3. Make and destroy gates according to need

    1. Increase the rate of transcription/translation of the rate limiting step or change its rate of degradation

    2. Bring in a new set of gates when you need them

Rate Limiting Factors in Catabolism:

  • Changes under different conditions

    • Fed, starved, resting, exercising, nutrients consumed etc.

Anything in blue (dark or light) or red could be RLS

  • Don’t regulate every step:

    • Can regulate enzyme activity or the availability of cofactors

PFK Enzyme – phosphofructokinase

  • Does not like a high concentration of its’ substrate, ATP

  • Does not have a high affinity for ATP

  • Activity of PFK is low at 5 mmol (the baseline concentration of ATP)

  • Once AMP is added, activity is increased dramatically

  • Through allosteric activation:

    • Activator binds allosterically (at a site away from the active site) on the enzyme, activates it, changes the shape of it to give it a higher affinity for its’ substrate

    • This is how AMP impacts phosphofructokinase

  • PFK also binds citrate allosterically, changes the shape of the active site to switch it off

  • Citrate inhibits PFK

  • Lots of citrate = lots of acetyl-CoA

  • Citrate can go back out into the cytoplasm and tell PFK to switch glycolysis off – allosteric inhibition

Hexokinase Enzyme – Feedback Inhibition

  • First step in glycolysis, traps glucose in the cytoplasm by phosphorylating it to creat GP6 (adds a phosphate group) using ATP

  • Inhibition by not using the product (GP6) prevents excessive trapping

    • Prevents ATP waste

    • Allows glucose to not go back out the cell

  • As GP6 builds up in the cell, negative feedback inhibits hexokinase

    • If GP6 is not being used, more glucose is not being trapped

  • If GP6 is used in the next step o glycolysis, inhibition is relieved and more glucose is trapped in the cytoplasm.

Example – Glycolysis in Exercise:

At rest (Glycolysis inhibited):

  • Glucose → GP6 but since there is no exercise, GP6 build up occurs

  • Negative feedback inhibits hexokinase from trapping more glucose in the cytoplasm

  • Energy charge is quite high while resting – lots of ATP, not much AMP

  • High energy charge acts of PFK (phosphofructokinase) and is getting switched off due to minimal AMP – it is not very attracted to ATP

  • Pyruvate kinase is also being switched off (at the end of glycolysis) by the high energy charge – not converting much ATP

During Exercise (glycolysis stimulates):

  • Glucose → GP6 but GP6 is being used so inhibition of hexokinase is relieved

  • Hexokinase can start trapping more glucose as it comes into the cell

  • Energy charge is now higher in AMP than ATP – overall low energy charge

  • PFK is becoming switched on and has a higher affinity binding to ATP due to increased levels of AMP – AMP allosterically activates PFK to switch on glycolysis

  • Feedforward stimulation from fructose 1,6-bisphosphate to pyruvate kinase to increase activity

    • feedforward stimulation would be like people at back of queue shouting for person at the front to hurry up

  • In high FAO (fatty acid oxidation),

    • High [citrate] = lots of acetyl-coa entering the krebs cycle

    • If citrate goes out into cytoplasm, it will switch of glycolysis by signalling PFK and pyruvate kinase that there are enough Kreb cycle intermediates

PDH (Pyruvate dehydrogenase) – covalent modification NOT allosteric inhibition/activation:

  • Inactivated entirely by phosphorylation

  • Covalent attachment of phosphate, catalysed by PDH kinase

  • Total amount of enzyme doesn’t change – just the ratio of phosphorylated to dephosphorylated

  • Reactivation by phosphate PDH phosphotase – release of phosphate = totally on

  • PDH activity a balance between kinase and phosphate

  • Kinases use ATP to add a phosphate to their substrate

  • High level of acetyl coa activates PDH kinase which adds a phosphate group to inactivate PDH

  • Insulin acts on PDH phosphatase to remove the phosphate from PDH to activate it again

The RLS in Glycolysis, β-oxidation, Krebs?

  • Could be availability of substrate (generally not)

  • Cell membrane transport and trapping

  • Mitochondria transport

    • Carintine availability

  • Oxidative capacity

    • Total activity of enzymes

    • Supply of oxygen (Electron transport chain – oxygen as the terminal electron acceptor)

  • It will depend on the circumstances

    • Overarching is cofactor availability (NAD+, FAD, CoA) an AMP level

    • Which itself is dictated by demand for ATP

Catabolism vs Anabolism:

  • Glycolysis vs gluconeogenisis

  • β-oxidation vs FA Synthesis

  • When one pathway is stimulated, the opposing pathway is inhibited

  • When they both occur at the same time it is a futile cycle

  • Not going to be breaking something while you’re also making it

  • Not always true – e.g. specialised tissues

Citrate as an Example:

  • Citrate is the first product in the krebs cycle

  • When citrate makes its way to the cytoplasm because there are enough Krebs cycle intermediates, citrate switches off glycolysis and on gluconeogenisis

  • Citrate switches of β-oxidation and on FA Synthesis

  • Balance of catabolic and anabolic pathways