Cell signalling- Richard

Question 1

Catabolism

Answer 1

Energy releasing, carbon-oxidising, degradative

Question 2

Anabolism

Answer 2

Energy-storing, biosynthetic

Question 3

Futile cycle

Answer 3

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

Question 4

Why is control important?

Answer 4

Ensuring only one cycle is active at one time

Question 5

Control can occur either internally or externally. True or false?

Answer 5

True `

Question 6

Internal control

Answer 6

participants in the pathway itself affect how it progresses

Question 7

External control

Answer 7

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],

Question 8

Biochemical reaction

Answer 8

Substrates, enzymes, products

Question 9

Explain internal control.

Answer 9

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.

Question 10

What is internal control also known as?

Answer 10

‘Feedback’

Question 11

What is negative feedback?

Answer 11

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

Question 12

What is the reverse of negative feedback?

Answer 12

Positive feedback- stimulation

Question 13

What is positive feedback?

Answer 13

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’)

Question 14

Feedback inhibition/precursor activation occurs via by 1 of 2 ways?

Answer 14

1. Indirect allosteric mechanisms
2. Direct mechanisms

Question 15

What is allosteric control also known as?

Answer 15

‘Non-competitive inhibition’

Question 16

Explain allosteric control.

Answer 16

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.

Question 17

What is one of the most common forms of allosteric control in metabolism?

Answer 17

ATP/AMP

Question 18

Give examples of allosteric control mechanisms:

Answer 18

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.

Question 19

What is Direct control also known as?

Answer 19

Competitive inhibition

Question 20

What is direct inhibition?

Answer 20

an inhibitor competes with substrate for the active site.

Question 21

Explain using an example direct inhibition.

Answer 21

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!

Question 22

Why is Direct inhibition competitive?

Answer 22

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.

Question 23

What is feedback inhibition?

Answer 23

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.

Question 24

Analysis of Enzyme Kinetics..

Answer 24

Velocity of a catalysed reaction can be determined by measuring amount of substrate consumed over time.

Question 25

What does comparison of reaction velocity versus substrate concentration give?

Answer 25

saturation of velocity at high substrate concentrations. This data allows derivation of Michaelis-Menton equation: V = Vmax.([S]/[S] + [Km])

Question 26

Lineweaver-Burke Plots (see 6th Feb practical).

Answer 26

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

Question 27

Explain the Lineweaver-Burke Plots

Answer 27

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.

Question 28

What is the more relevant calculations?????

Answer 28

Lineweaver-Burke Plots

Question 29

Concept of Affinity- applies to both receptor binding and enzyme kinetics:

Answer 29

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!

Question 30

External control:

Answer 30

Other factors from elsewhere (eg. hormones, nerve impulses, etc – collectively known as SIGNALS) affect reaction/pathway’s progress.

Question 31

External control can occur over a range of mechanisms. What are they?

Answer 31

 Protein/Enzyme Activity  Gene Expression  Membrane Permeability

Question 32

Explain external control using an example.

Answer 32

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.

Question 33

Definition of Signal Transduction:

Answer 33

A basic process in molecular cell biology involving the conversion of a signal from outside the cell to a functional change within the cell.

Question 34

Amino Acid-derived Hormones

Answer 34

Can’t cross PM; ligands for (ie. bind to) membrane receptors Initiate intracellular responses  Gene expression/protein activity/memb perm

Question 35

Steroid hormones

Answer 35

Hydrophobic; cross PM Ligands for (ie. bind to) cytoplasmic receptors Complex binds to DNA (“Zn fingers”)  Gene expression

Question 36

How many hormone receptors are there and what are they?

Answer 36

2- Non-steroid hormone receptors Steroid hormone receptors

Question 37

What are non-steroid hormone receptors?

Answer 37

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.

Question 38

Steroid hormone receptors…

Answer 38

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.

Question 39

Analysis of Receptor Binding (similar to Michaelis-Menton):

Answer 39

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).

Question 40

Scatchard Plot

Answer 40

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.

Question 41

IC50(or Ki): measure of inhibitor/antagonist affinity:

Answer 41

“concentration of inhibitor necessary to halve the reaction rate of an enzyme-catalysed reaction

Question 42

EC50: measure of activator/agonist affinity:

Answer 42

“concentration of agonist that induces response halfway between baseline & maximal response”. Both are used with ‘dose-response curves’

Question 43

Definition of ‘Ligand’; could encompass:

Answer 43

Enzyme/substrate Enzyme/inhibitor Receptor/signal (hormone, cytokine, agonist, etc) Receptor/blocker (inhibitor, antagonist, etc)

Question 44

Links to pharmacology aspect of this module (& to pharmaceutical science industry).

Answer 44

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!)

Question 45

Cellular response A:

Answer 45

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)

Question 46

Covalent modification:

Answer 46

adding chemical gps to proteins to alter their activity

Question 47

Phosphorylation:

Answer 47

occurs on Ser/Thr (>99%), or Tyr (<1%) residues.

Question 48

Methylation:

Answer 48

attachment of methyl groups to proteins

Question 49

Myristoylation Palmitoylation, Oleoylation

Answer 49

attachment of Fatty Acids to protein (often tethers proteins to membranes).

Question 50

Glycosylation:

Answer 50

attachment of sugars to proteins (eg CD45)

Question 51

Conformation [shape] dictates Function, therefore….

Answer 51

Phosphorylation [change in conformation] causes Change in Function [ie. a response!]

Question 52

Receptor Tyr Kinases (RTKs): many different sub-families-

Answer 52

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)

Question 53

Signalling Cascades:

Answer 53

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)

Question 54

Practical session:

Answer 54

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.

Question 55

Aims of Thursday’s practical:

Answer 55

[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.