Cell signalling- Richard Flashcards
Catabolism
Energy releasing, carbon-oxidising, degradative
Anabolism
Energy-storing, biosynthetic
Futile cycle
glycolysis & gluconeogenesis exact opposites-> so to have both going ‘at full speed’ all the time would simply expend a lot of energy without achieving anything
Why is control important?
Ensuring only one cycle is active at one time
Control can occur either internally or externally. True or false?
True `
Internal control
participants in the pathway itself affect how it progresses
External control
factors from elsewhere affect the pathway’s progress eg. hormones, availability of foodstuffs/energy, conscious decisions whether to act or not [ie. nervous impulses sent by the brain],
Biochemical reaction
Substrates, enzymes, products
Explain internal control.
Because enzymes are themselves proteins, any biochemical changes to enzymes (ie. inhibition or activation) will affect their ability to catalyse their reactions.
eg. Addition or removal of phosphate groups from enzymes (ie. phosphorylation or dephosphorylation) is an important controlling factor.
What is internal control also known as?
‘Feedback’
What is negative feedback?
inhibition of a process by presence of large amounts of final Product (‘sending the message that the process has produced enough product’) eg. Hexokinase is inhibited by Glucose6-P
What is the reverse of negative feedback?
Positive feedback- stimulation
What is positive feedback?
Precursor/Reactant Activation – activation of a process by presence of large amounts of initial reactant (‘sending the message that the process hasn’t consumed enough reactant’)
Feedback inhibition/precursor activation occurs via by 1 of 2 ways?
- Indirect allosteric mechanisms
- Direct mechanisms
What is allosteric control also known as?
‘Non-competitive inhibition’
Explain allosteric control.
A regulator molecule (not the substrate) binds to the enzyme at a different site than the one to which the substrate binds.
*Regulator binding alters the enzyme’s conformation so that its activity is changed.
What is one of the most common forms of allosteric control in metabolism?
ATP/AMP
Give examples of allosteric control mechanisms:
Eg. the glycolytic [catabolic] enzyme PFK is
Allosterically inhibited by ATP/stimulated by AMP
while the gluconeogenic [anabolic] enzyme PEPCK is
Allosterically stimulated by ATP/inhibited by AMP.
What is Direct control also known as?
Competitive inhibition
What is direct inhibition?
an inhibitor competes with substrate for the active site.
Explain using an example direct inhibition.
Eg. succinic dehydrogenase, which catalyzes the oxidation of succinic acid (Fig a) to produce fumaric acid (as part of the TCA Cycle).
Malonic acid inhibits the enzyme: the structure of malonic acid allows it to bind to the same site on the enzyme (Fig b). But in this case, the reaction is NOT catalysed!
Why is Direct inhibition competitive?
The inhibition is ‘competitive’ because if you increase the ratio of succinic acid to malonic acid in the mixture, you will gradually restore the rate of catalysis – succinic acid and malonic acid compete for the enzyme’s active site.
What is feedback inhibition?
the product of an entire pathway may compete with substrates at an enzyme’s active site, & so inhibit the catalysis of an ‘upstream’ reaction early in the pathway.
Analysis of Enzyme Kinetics..
Velocity of a catalysed reaction can be determined by measuring amount of substrate consumed over time.
What does comparison of reaction velocity versus substrate concentration give?
saturation of velocity at high substrate concentrations.
This data allows derivation of Michaelis-Menton equation:
V = Vmax.([S]/[S] + [Km])
Lineweaver-Burke Plots (see 6th Feb practical).
Plotting the reciprocals of Michaelis-Menton data provides a more precise way to determine Vmax and Km: 1/V=Km/Vmax.1/[S] + 1/Vmax
AKA y=mx+c
Explain the Lineweaver-Burke Plots
1/V=y-axis variable
1/[S]=x-axis variable
Vmax= reciprocal of intercept point with the y axis
Km/Vmax = slope of the graph.
What is the more relevant calculations?????
Lineweaver-Burke Plots
Concept of Affinity- applies to both receptor binding and enzyme kinetics:
Km: concentration of ligand/substrate that induces 50% maximal binding to enzyme.
Importantly, the higher the affinity, the LESS ligand is needed to induce 50% maximal binding, so HIGH-AFFINITY INTERACTIONS HAVE LOW Km VALUES!
External control:
Other factors from elsewhere (eg. hormones, nerve impulses, etc – collectively known as SIGNALS) affect reaction/pathway’s progress.
External control can occur over a range of mechanisms. What are they?
Protein/Enzyme Activity
Gene Expression
Membrane Permeability
Explain external control using an example.
Receptor [eg. IR] is a PM enzyme that has Protein Kinase enzymatic activity in its cytosolic domain.
On receptor occupancy (when signal arrives at external surface of cell), conformation of receptor’s cytosolic domain changes, enabling it to activate substrate proteins [eg. IRS] (usually via phosphorylation). These phosphorylated proteins then initiate the cellular response.
Definition of Signal Transduction:
A basic process in molecular cell biology involving the conversion of a signal from outside the cell to a functional change within the cell.
Amino Acid-derived Hormones
Can’t cross PM; ligands for (ie. bind to) membrane receptors
Initiate intracellular responses
Gene expression/protein activity/memb perm
Steroid hormones
Hydrophobic; cross PM
Ligands for (ie. bind to) cytoplasmic receptors
Complex binds to DNA (“Zn fingers”)
Gene expression
How many hormone receptors are there and what are they?
2-
Non-steroid hormone receptors
Steroid hormone receptors
What are non-steroid hormone receptors?
are integral transmembrane proteins.
-Extracellular regions interact with hormones.
-Series of repeated stretches of hydrophobic Aas (Represent memb-spanning helices “T-M domains”)
-Cytosolic regions interact with transducers, & hence initiate intracellular responses to signals.
Steroid hormone receptors…
consist of dimers of at least 3 functional modules or domains:
the domain responsible for binding hormone (and also the second unit of the dimer).
the zinc-finger domain needed for DNA binding (to the steroid response element [SRE]).
a domain needed for the receptor to activate the promoters of the genes being controlled.
Analysis of Receptor Binding (similar to Michaelis-Menton):
Saturation Binding: as hormone concn rises, concn of hormone Bound to receptor rises until no more can bind. Affinity of binding can be expressed using Dissociation Constant “Kd“ (concentratn of signal/ligand that induces 50% maximal binding).
Scatchard Plot
mathematical manipulation allows saturation binding data to be expressed as straight line. [Hill Co-efficient – alternative analysis.]
Slope of line = -1/Kd; intercept on X-axis = density of receptors.
IC50(or Ki): measure of inhibitor/antagonist affinity:
“concentration of inhibitor necessary to halve the reaction rate of an enzyme-catalysed reaction
EC50: measure of activator/agonist affinity:
“concentration of agonist that induces response halfway between baseline & maximal response”.
Both are used with ‘dose-response curves’
Definition of ‘Ligand’; could encompass:
Enzyme/substrate
Enzyme/inhibitor
Receptor/signal (hormone, cytokine, agonist, etc)
Receptor/blocker (inhibitor, antagonist, etc)
Links to pharmacology aspect of this module (& to pharmaceutical science industry).
NB. ‘Drugs’ are invariably synthetic molecules designed & manufactured to mimic natural molecules in fulfilling the above roles, & hence influencing the progress of reactions within the cell/body (& impacting on patient/client health!)
Cellular response A:
On arrival of a signal (and subsequently a signal transduction event) intracellular proteins are activated to initiate a response
- 2nd messengers released into vicinity of enzymes. eg. cAMP/Protein Kinase A
*Zymogenic activation.
–Proteases cleave immature to mature enzyme (eg. Trypsinogen trypsin)
Covalent modification:
adding chemical gps to proteins to alter their activity
Phosphorylation:
occurs on Ser/Thr (>99%), or Tyr (<1%) residues.
Methylation:
attachment of methyl groups to proteins
Myristoylation Palmitoylation, Oleoylation
attachment of Fatty Acids to protein (often tethers proteins to membranes).
Glycosylation:
attachment of sugars to proteins (eg CD45)
Conformation [shape] dictates Function, therefore….
Phosphorylation [change in conformation] causes Change in Function [ie. a response!]
Receptor Tyr Kinases (RTKs): many different sub-families-
Receptor occupancy dimerization & autophosphorylation.
2.Attraction of SH2 domains; binding to substrates (phosphorylated by RTK) and adaptors (not phosphorylated; instead, facilitate phosphorylatn of substrates).
3.Substrates initiate response (often via multi-step ‘cascade’ process):
–Phosphorylate several types of ‘downstream’ proteins ‘signalling cascades’.
–Phosphorylate ‘end-target’ proteins phosphorylatn results in a conformational change that is itself a response (eg. STAT3 moves to nucleus & acts as Transcription Factor)
Signalling Cascades:
Kinases can phosphorylate other kinases, which can phosphorylate other kinases, & so on.
*Tyr Kinases (comparatively small in number; mostly membrane-bound RTKs - eg.InsR)
*Ser/Thr Kinases (numerous [>99%]); mostly cytosolic - eg. PKA)
Practical session:
This experiment will focus on the hydrolysis of phenolphthalein diphosphate by alkaline phosphatase (ALP), & the impact of vanadate (a phosphatase inhibitor) on the affinity of alkaline phosphatase for its substrate.
*Mellgren JBC 252 p6082: ALP catalyses dephosphorylation of phospho-proteins (& other phosphorylated substrates: eg. ATP, phenolphthalein diphosphate).
–Dephosphorylation of phospho-proteins can impact on signal transduction. Passing on of a signal (& hence initiation of a response) depends on protein kinase-catalysed phosphorylation, & dephosphorylation provides a means of rapid reversibility of phosphorylation: signals can be terminated rapidly after they’re initiated (ie they provide the ‘off’ part of the signal!)
Clinical relevance of practical: When the liver is damaged, ALP may leak into the bloodstream. High levels of ALP can indicate liver disease or bone disorders.
Aims of Thursday’s practical:
[i] use experimental data to determine the Km (& hence affinity) of Alkaline Phosphatase for its substrate. [ii] investigate impact of inhibitor (vanadate) on ALP’s affinity for its substrate.
