Regulation of Metabolism Flashcards
Why does metabolism require control?
- To co-ordinate metabolic processes
- Vital that some of the enzymes involved in these processes are subject to strict controls
What are 4 factors that may be involved in the regulation of biochemical pathways?
- Concentrations of substrates and products
- Modifications
- Endocrine signals
- Other enzymes
General Mechanisms of Enzyme Regulation: What did Leonor Michaelis and Maud Menten propose?
- That the enzyme reversibly combines with its substrate
- To form an ES complex that subsequently breaks down to product, regenerating free enzyme
What does the Michaelis-Menten equation describe?
Describes how reaction velocity varies with substrate concentration
What is the Michaelis-Menten equation for the formation of an ES complex?
What is the Michaelis-Menten equation for how velocity varies with substrate concentration?
What are the assumptions of the Michaelis-Menten Equation (3 points)
- [S] is small, much greater than [E], so that the amount of substrate bound by the enzyme at any one time is small.
- [ES] does not change with time as it is in ‘steady state’
- Only initial velocities are used in analysis of enzyme reactions, as it is only at this time when the reaction is linear with time
What is The Michaelis constant
Km
What is Km characteristic of and what does it reflect?
- characteristic of an enzyme and its substrate
- reflects the affinity of the enzyme for that substrate
What is Km numerically equal to?
Numerically equal to [S] at which the reaction velocity is at ½Vmax
Km does NOT vary with with what?
Km does not vary with the concentration of enzyme
What does a numerically small Km (low) reflect?
- reflects a high affinity of the enzyme for substrate
-because a low concentration of substrate is needed to half-saturate the enzyme - i.e. to reach ½Vmax.
- Enzymes like this are usually targeted for regulation
What does a numerically large Km (high) reflect?
- reflects a low affinity for the substrate
- because a high concentration is required to half-saturate the enzyme
- Enzymes like this are not usually targeted for regulation
What is the rate of reaction for the Michaelis-Menten Equation directly proportional to?
The enzyme concentration [S]
What is the order of reaction for enzyme regulation?
When [S] is much less than Km:
-The velocity of the reaction is roughly proportional to [S]
-The rate is said to be first order with respect to substrate
When [S] is much greater than Km:
- The velocity is constant and equal to Vmax
- The rate of reaction is then independent of [S]
- Is said to be zero order with respect to substrate concentration.
Allosteric Modification
Some molecules may bind non-covalently to an enzyme…. What does this result in and what are these molecules called?
- Inhibits the activity of said enzyme
- Molecules that do this are called effectors
What are the two types of effectors?
- Homotropic Effectors
- Heterotropic Effectors
What are the main characteristics of Homotropic Effectors?
- When the substrate is an effector
- They are usually positive
- Significance: Km is decreased several fold for a small increase in [S].
What are the main characteristics of Heterotropic Effectors?
- The effector is not the substrate
- May have a stimulatory or inhibitory effect
What is a very rapid (instantaneous) form of regulation?
Allosteric modification
What does induction and Repression of Synthesis represent?
- Represents adaptive regulation - whereby enzyme synthesis is either enhanced or decreased by certain physiological situations
- Slow (days) mechanism of regulation
How are the processes of glycogen synthesis and glycogen breakdown reciprocally regulated
- allosterically
- covalently
- induction/repression of synthesis
What are the key regulatory enzymes in glycogen metabolism?
SYNTHESIS: Glycogen Synthase
BREAKDOWN: Glycogen Phosphorylase
Fed state of glycogen metabolism:
Glucose converts to? ——->
Glucose—–>Glycogen
Fasted state of glycogen metabolism:
Glycogen converts to? ——->
Glycogen—–>Glucose
What does glucose 6-phosphate activate and inhibit in glycogen metabolism (allosteric regulation)
Glucose 6-phosphate….
Activates:Glycogen Synthase
Inhibits:Glycogen phosphorylase
Allosteric regulation:
What is AMP and how is it generated?
- AMP= Adenosine Monophosphate
- AMP is generated when skeletal muscle uses ATP to contract
AMP has no effect on glycogen synthase but does what instead?
Allosterically activates Glycogen phosphorylase – very important in exercising skeletal muscle
Allosteric regulation:
What does a high concentration of ATP mean?
- Means the energy status of the cell is very high
- Hence there is no requirement to breakdown Glycogen
ATP has no effect on glycogen synthase but does what?
- ATP allosterically inhibits Glycogen phosphorylase
In a fed state, there is an increase of glucose concentration.
So there is no need for what?
- no need to continue breaking down glycogen for glucose production
What does glucose allosterically inhibit?
- Allosterically inhibits glycogen phosphorylase
- with no effect on Glycogen Synthase
-very important for the liver
Allosteric regulation of enzymes is classified as what type of regulation?
‘Within the cell’ regulation
Allosteric regulation involves the binding of what? and what does this result in?
- Involves the binding of an effector molecule to the enzyme
-Thereby altering its activity
Covalent regulation of metabolic processes is usually in response to what? and give an example.
An extracellular stimulus
- Such as from a hormone which then binds to an extracellular receptor
- leads to a cascade of biochemical events leading to an effect on the target enzymes
What are the 6 components of hormone action to consider in general terms, before we can look at what happens to glycogen synthase and glycogen phosphorylase?
- Signal
- Receptor
- Coupling
- Amplification
- Effect
- Termination
Hormones: 1=signal
What are the 3 categories of hormones?
- Peptides or polypeptides
- Steroid hormones
- Amino acid derivatives
Hormones: 2= The receptor
What are hormones unable to stimulate?
Hormones cannot stimulate a metabolic process directly
What do hormones have to bind to in the first instance? And give 2 examples.
- Have to bind to a specific receptor
Steroids:
- Intracellular receptors interact with chromatin and effect mRNA transcription – not discussed further
Others:
– Bind to receptors located on the external surface of the plasma membrane
Extracellular receptors are usually what?
Glycoprotiens
N-linked region of extracellular receptors contain oligosaccharides on the extracellular surface: What do these convey?
– these convey specificity
Receptor distribution is dependent on what?
– dependent upon the tissue
Hormones: 3= Coupling and G proteins
Binding of a hormone to its receptor is not sufficient on its own to affect what?
Metabolic pathways
What is hormone receptor binding coupled to?
Hormone-Receptor binding is coupled to:
- An intracellular event
Peptide and amino acid hormone receptors are coupled to what? and where is it found?
- A specific ‘Guanyl-stimulatory binding protein’ (GS-protein)
- Found on the intracellular surface of the plasma membrane
How many subunits does GS consist of and what are they?
GS consists of 3 subunits:
α subunit (45 kDa)
β subunit (35 kDa)
γ subunit (7kDa)
Hormones: 3= Coupling and G proteins
α-subunit can interconvert between what 2 forms? and what does this depend on?
- a form which binds GDP
- a form that binds GTP
Depending on whether there is a hormone signal or not
When there is no hormone signal what does the α-subunit do?
- The α-subunit ‘rests’ in the GDP binding form
- There is no interaction between the unoccupied receptor and GS-protein
Hormones: 3= Coupling and G proteins
When there is a hormone signal, the α-subunit does what?
- changes its conformation and loses the GDP and instead binds GTP
Fill in the blanks:
The α-GTP subunit ——— from the ———— and binds to and ————-, an enzyme called ———- which is located within the ———–.
- Dissociates
- Rest of the GS-protein
- Activates
- Adenylate cyclase
- Plasma membrane
The α-GTP subunit dissociates from the rest of the GS-protein and binds to and activates an enzyme called adenylate cyclase which is located within the plasma membrane.
In the absence of further hormone stimulation , what happens?
- The GTP is hydrolysed to GDP
- And the α-GDP subunit dissociates from adenylate cyclase and re-associates with the rest of the GS-protein subunits
The βγ subunit does not undergo what type of change? and what does it do instead?
-The βγ subunit does not undergo a conformational change
- Acts as its own signalling molecule activating and inhibiting various enzymes
Hormones: 4= Amplification and cAMP
When bound to the α-GTP subunit of the GS-protein, adenylate cyclase is activated and is able to do what?
- Able to catalyse the formation of cyclic AMP (cAMP) from ATP.
Hormones: 4= Amplification and cAMP
Hundreds of molecules of cAMP are produced for every activated molecule of adenylate cyclase. What is the term used to describe this?
– this is the amplification of the hormone signal
Hormones: 5=Effect by protein kinases
What is cyclic AMP called?
Cyclic AMP is called a second messenger
Hormones: 5=Effect by protein kinases
cAMP is a potent activator of another enzyme, what is it?
– Protein Kinase A
cAMP-dependent protein kinase
Protein Kinase A is a……??
And is made up of what?
Protein Kinase A is a tetramer
- 2 regulatory subunits
- 2 catalytic subunits
What do the two free catalytic subunits do?
- They catalyse the phosphorylation of specific serine or threonine residues on target proteins
Phosphorylated proteins may do what 3 things?
- Activate enzymes
- Inactivate enzymes
- Modulate the activity of cellular ion channels
Protein kinase A can phosphorylate what? and what does this cause?
-Can phosphorylate specific proteins that bind topromoter regions of DNA
- Causing increased expression ofspecific genes
Hormones: 6=Termination
What happens during termination?
- Loss of hormone signal
- Dephosphorylation of proteins
- Hydrolysis of cAMP
Explain what happens in the process of dephosphorylation of proteins.
- The phosphate groups added to proteins by protein kinases areremoved
-They are removed by the actions of phosphoprotein phosphatases: enzymesthat hydrolytically cleave phosphate esters
- This ensures that changes in enzymatic activity induced by proteinphosphorylation are not permanent
Hydrolysis of cAMP:
cAMP is readily hydrolysed to what?
cAMP is readily hydrolysed to 5’-AMP byphosphodiesterase.
5’-AMP is not what type of molecule?
5’-AMP is not an intracellular signalling molecule
What is the target enzyme in the Inhibition of synthesis by a cAMP cascade?
Target enzyme = GlycogenSynthase
Inhibition of synthesis by a cAMP cascade:
Glycogen synthase exists in what 2 forms? and give key features of each
- “A” form:
- NOTphosphorylated
- Is the most activeform - “B” form:
- ISphosphorylated
- Inactive
Inhibition of synthesis by a cAMP cascade:
Glycogen synthase is converted from A to B form by what?
By phosphorylation’s ata numberofsites on theenzyme.
After the conversion from A to B form the level of inactivation is proportional to what?
It’s degree ofphosphorylation
Inhibition of synthesis by a cAMP cascade:
What 3 things result in the activation of adenylate cyclase?
- Binding of the hormone glucagon to hepatocyte receptors
- Binding of the hormone adrenaline tohepatocyte receptors
- Binding of adrenaline to muscle cell receptors
Inhibition of synthesis by a cAMP cascade:
cAMP is synthesised, what does this activate?
- Activates protein kinase A
After protein kinase A is activated, what does this result in protein kinase A doing?
- Protein kinase A phosphorylates glycogen synthase A toglycogen synthase B
- Therefore inactivates glycogensynthesis
Glycogen synthase B can be transformed back to glycogen synthase A by what?
- By removing the phosphate groups hydrolytically using phosphoprotein phosphatase type 1
– Activation of breakdown by a cAMP cascade
The binding of glucagon or adrenaline to receptors, signalsthe need for what?
Signalsthe need for glycogen to be degraded:
– Either to elevateblood glucose levels (contributed by liver glycogen)
- Or toprovide energy in exercising muscle
Activation of breakdown by a cAMP cascade
What 3 things are activated during this process?
- Activation of protein kinase A
- Activation of phosphorylase kinase
- Activation of glycogen phosphorylase
Activation of breakdown by a cAMP cascade:
How is phosphorylase kinase activated?
- Active protein kinase A phosphorylates the inactive form ofphosphorylase kinase, resulting in its activation
Activation of breakdown by a cAMP cascade:
Explain the process of the activation of glycogen phosphorylase.
- Glycogen phosphorylase exists in an inactive “B” form and an active“A” form
- Active phosphorylase kinase, phosphorylates glycogen phosphorylaseb.
- Converting it into active glycogen phosphorylase A, which beginsglycogen breakdown.
- Phosphorylase A is reconverted to phosphorylase B. Byphosphoprotein phosphatase type 1
Summary of the reciprocal regulation of glycogen synthesis and degradation:
Glycogen synthesis and degradation are both regulated by the same what?
Both regulated by the same hormonal signals
Summary of the reciprocal regulation of glycogen synthesis and degradation:
What do the following things result in:
- Elevated insulin levels
- Elevated Glucagon/Adrenaline levels
- Elevated insulin level=overall increased glycogen synthesis
- Elevated glucagon/adrenaline=cause increased glycogen degradation
Summary of the reciprocal regulation of glycogen synthesis and degradation:
What do cyclic AMP levels fluctuate in response to?
Fluctuate in response to hormonal stimuli
Summary of the reciprocal regulation of glycogen synthesis and degradation:
What do cAMP levels in cells increase and decrease in response to?
cAMP levels increase=in response to hormonal stimuli
cAMP levels decrease= in the presence of insulin
Summary of the reciprocal regulation of glycogen synthesis and degradation:
Give an example of hormonal stimuli that would increase cAMP levels.
- Glucagon and adrenaline in liver
- Adrenaline in muscle
Summary of the reciprocal regulation of glycogen synthesis and degradation:
What are key enzymes phosphorylated by?
A family of kinases
Only some of which are cAMP dependent
Summary of the reciprocal regulation of glycogen synthesis and degradation:
What does phosphorylation of an enzyme cause?
- Causes a conformational change
- That affects the active site
- And can greatly increase or decrease the catalytic activity of some enzymes
Phosphorylase kinase can also be activated how?
- Allosterically in muscle
Phosphorylase kinase can also be activated allosterically in muscle. Step 1:
Muscles contract because of the release of what?
- Release of Ca2+ release
- From sarcoplasmic reticulum
Phosphorylase kinase can also be activated allosterically in muscle. Step 2:
Ca2+ binds to a subunit of phosphorylase kinase called what?
Calmodulin
Phosphorylase kinase can also be activated allosterically in muscle. Step 3:
Ca2+-Calmodulin activates phosphorylase kinase: This thereby activates what?
The glucose release from this then fuels what process?
- Thereby activating glycogen phosphorylase and hence causing glycogen breakdown
- Glucose released then fuels muscle contractions
Phosphorylase kinase can also be activated allosterically in muscle. Step 4:
When muscle relaxes, Where does Ca2+ return to?
What then happens to the phosphorylase kinase and glycogen phosphorylase A?
- Ca2+ returns to the sarcoplasmic reticulum
- Phosphorylase kinase=becomes inactive
- Glycogen phosphorylase A = converted to the inactive, glycogen phosphorylase B
