Control of metabolism

What is the protein concentration inside cells?

- (0.2-0.3 g/ml in the cytosol and up to 0.5 g/ml in the mitochondrial matrix.

What is the abundance of different kinases and phosphatases?

- 100,000 phosphorylation sites, ~99% of which are on serine and threonine residues and ~1% on tyrosine residues. - 428 serine/threonine kinases and 90 tyrosine kinases. - Therefore, the average Ser/Thr kinase would act on ~230 distinct residues and the average Tyr kinase on ~12. - There are only 147 protein phosphatases; of these, only 40 dephosphorylate phospho-serine and -threonine. -There are an identical number of Tyr kinases and phosphatases and therefore each Tyr phosphatase acts on only ~12 - Each Ser/Thr protein phosphatase would on average dephosphorylate ~2500 different targets

What is the structure of HIF-1 and its function?

- A critical response to hypoxia (low oxygen) is the induction of the hypoxia-inducible transcription factor, HIF-1, a basic helix-loop-helix transcription factor ( contains HIF-1α and HIF1-1β subunit) - HIF-1 increases the expression of genes that encode glycolytic enzymes.

What is the futile cycle of glycolysis and what is its function?

- A futile cycle, also known as a substrate cycle, occurs when two metabolic pathways run simultaneously in opposite directions and have no overall effect other than to dissipate energy in the form of heat. - Futile cycles may have a role in metabolic regulation, where a futile cycle would be a system oscillating between two states and very sensitive to small changes in the activity of any of the enzymes involved.. - The cycle does generate heat, and may be used to maintain thermal homeostasis

How does phosphorylation increased sensitivity (ultra-senstivity).

- A small change in kinase or phosphatase VMAX causes a much larger change in the proportion of phosphorylated substrate when both enzymes are operating close to saturation with their protein substrate. . - A much less sensitive system results if the kinase and phosphatase are not saturated. - If the kinase and phosphatase are regulated in opposite directions then we get even greater sensitivity; e.g. PKA phosphorylates and activates phosphorylase kinase and inhibits PP1 by phosphorylation of the G subunit and inhibitor-1.

How is the rate of glycogen breakdown increased during muscle contraction?

- AMP production increases rate of phosphorylase, increasing the concentration of glucose 1-P

What is a positive regulator of PFK?

- AMP which acts as an allosteric activator. - Produced during muscle contraction

How is hexokinase positively regulated during muscle contraction?

- AMP which increases the rate of PFK - PFK increases the rate of conversion of fructose 6-P into fructose 1, 6-bis P. - Decrease in concentration of fructose 1, 6-bis P decreases the concentration of glucose-6-P as both are in equilibrium. - Since glucose-6-P is an allosteric inhibitor of hexokinase, lowering its concentration increases its rate of activity.

What is a positive regulator of pyruvate kinase during muscle contraction?

- AMP which increases the rate of PFK - PFK increases the rate of conversion of fructose 6-P into fructose 1, 6-bis P. - fructose 1, 6-bis P acts as an allosteric activator of pyruvate kinase (feedforward)

How does insulin in the liver favour glycolysis?

- Activates a low Km phosphodiesterase that hydrolyses cAMP to 5'AMP which switched off PKA activity.

How is glycogen metabolism in the muscle controlled?

- Activation of adenylate cyclase by adrenalin leads to an increase in cAMP concentration, which activates protein kinase A (PKA) by promoting dissociation of the regulatory and catalytic subunits. - PKA phosphorylates a large number of enzymes including phosphorylase kinase. (αβγδ)4, phosphorylation of the α and β subunits) - Phosphorylase kinase phosphorylates phosphorylase b into phosphorylase a. This increases the rate of glycogen breakdown. - Phosphorylase kinase and PKA also phosphorylates glycogen synthase an into glycogen synthase b which inhibits glycogen synthesis.

What is the role of p53 in controlling flux through a normal cell?

- Activation of p53 inhibits glycolysis by activating TIGAR (TP53- induced glycolysis and apoptosis regulator). - TIGAR has fructose-2,6- bisphosphatase activity. Inhibition of the glycolytic pathway diverts flux into the pentose phosphate pathway, leading to NADPH production and protection against oxidative stress.

When is an enzyme most sensitive to change?

- An enzyme will be most sensitive to changes in substrate concentration when these occur at or below the KM. - However, even at these levels the response is only proportional - a doubling in substrate concentration will at most lead to an approximate doubling of rate.

How does 'The fly that can't fly' experiment prove demonstrate that glycolytic enzymes can associate with each other, with cytoskeletal components and with myofibrillar proteins?

- An isoform of (GPDH-1) is localised to the Z discs and M lines in Drosophila flight muscle. Localisation results in co-localisation of the glycolytic enzymes aldolase and GAPDH. - In muscles of strains carrying Gpdh null alleles, there was no localisation of GAPDH and aldolase, but localisation could be restored by the transformation of the mutants with plasmids expressing GPDH-1. - Localisation of GPDH-1 is due to a tripeptide at its C terminus. - Expression of the GDPH-3 isoform which lacks this tripeptide was itself not localised and failed to restore co-localisation of aldolase and GAPDH. These flies, which had nearly normal levels of these glycolytic enzymes, were incapable of flight. - Could prove local ATP production however GPDH-1 might just be playing a structural role.

What is the function of the liver in terms of carbohydrate metabolism.

- Buffers blood glucose levels, maintain blood glucose homeostasis. - The brain is dependent on glucose and requires a steady supply of glucose.

Describe the concerted model for cooperation.

- Concerted model for positive cooperativity. - Conformational change in one subunit is necessarily conferred to all other subunits. Thus, all subunits must exist in the same conformation. - The model further holds that, in the absence of any ligand (substrate or otherwise), the equilibrium favours one of the conformational states, T or R. The equilibrium can be shifted to the R or T state through the binding of one ligand (the allosteric effector or ligand) to a site that is different from the active site (the allosteric site). - Only explains positive cooperativity.

How can Compartmentation by binding be deduced?

- Considering the concentrations of the concentrations of their substrates. - For example in skeletal muscle, high Glyceraldehydephosphate dehydrogenase conc yet a low Glyceraldehyde-3P conc. - Suggests Glyceraldehyde-3P bound to proteins.

What is the direct link between regulation of the cell cycle and cell membrane?

- D-type cyclins and their catalytic binding-partner kinases (CDK4 or CDK6) are required for cell division. - They exhibit peak activity during early G1 phase, when the cell synthesises components needed for DNA replication and cell division. - Phosphorylation of PFK1 and PKM2 by a cyclin D3-CDK6 complex inhibits the activities of these glycolytic enzymes by promoting dissociation of the more active tetramers to the less active dimers, increasing the flow of glycolytic intermediates into the pentose phosphate (PP) and serine synthesis (SS) pathways

What can demonstrate the fast recruitment of GLUT4?

- Experiments with transgenic mice containing GLUT4 transporters fused to GFPs. - Using a window chamber on the skin of a mouth, light can be detected when GLUT4 is recruited.

How can you report local ATP concentrations with a cell?

- Firefly luciferase, which shows ATP-dependent (KM ~ 1 mM) luminescence, can be used as a probe for intracellular ATP concentrations. By targeting this enzyme to specific subcellular locations, it can be used to report on local ATP concentrations. - ATP conc at the plasma membrane near the mitochondria remain high

How is Substrate channelling observed with 1,3-bisphosphoglycerate?

- GAPDH white - PGK yellow - 1,3-bisphosphoglycerate does not diffuse away in a free unbound form but channels to the next enzyme. Doesn't get lost to competing reactions.

What are the different GLUT transporters and where are they found?

- GLUT1 and 3, present in nearly all mammalian cells, are responsible for basal glucose transport (KM ~ 1 mM, which is much less than the normal blood glucose level (4-8 mM)). - GLUT2, present in liver and pancreatic β-cells, has a high KM for glucose (15-20 mM) and hence uptake rate is proportional to the blood glucose level. - GLUT4 (KM ~ 5 mM) is recruited to the plasma membrane of muscle and fat cells by insulin, which thus promotes glucose uptake by these cells.

How does Glucagon effect the liver?

- Glucagon and β-adrenergic agonists enhance adenylate cyclase activity, leading to increased cAMP concentration, activation of PKA and phosphorylation of pyruvate kinase and fructose-2,6-bisphosphatase at serine residues. - Produces coordinate regulation.

What liver enzyme relies on substrate level control?

- Glucokinase (hexokinase isozyme), has a relatively large Km compared to hexokinase found in muscles. - Glucose converted to glycogen at high blood glucose concentrations (after meals)

In the liver, what regulates the a form of phosphorylase when blood glucose levels are high?

- Glucose binding. - Binding of glucose shifts the T ↔ R equilibrium to the T form and promotes dephosphorylation of Ser14 by PP1, leading to inactivation (b form). T - Thus, when blood glucose concentration is high, further production of glucose by liver glycogenolysis is inhibited. - High levels of blood glucose also activate glycogen synthesis. - Conversion of phosphorylase a to phosphorylase b leads to the release of PP1, which binds tightly to the a form and is then free to activate glycogen synthase

What are the enzymes which carry out muscle glycogen breakdown and synthesis?

- Glucose-1-phosphate to uridine diphosphate glucose is exergonic and so reaction is favoured to the right.

During anaerobic respiration, why does the glucose produced from glycogen yield a greater amount of net ATP than glucose coming in from the bloodstream?

- Glycogen is broken down into glucose-6-p, doesn't need to be phosphorylated by ATP to enter glycolysis.

What are the three pathways of glucose metabolism?

- Glycolysis - Glycogen breakdown and synthesis - Gluconeogenesis

What genes do HIF-1 turn on?

- HIF-1 turns on glycolysis and at the same time turns off flux into the TCA cycle. - HIF-1 induces the PDK1 gene, producing a protein kinase that phosphorylates the E1 subunit of pyruvate dehydrogenase (PDH) and inactivates it.

What are the advantages of Substrate channelling?

- High fluxes with low intermediate concentrations (no need to diffuse) - Isolation of intermediates from competing reactions - Protection of unstable intermediates (G3AP and DHAP) - Circumvention of the effects of unfavourable equilibria (increased local concentration of substrates) - Faster response/reduction of lag times in transients between steady states (intermediates don't have to diffuse away and find the active site) - Regulation of flux

How is Substrate channelling observed with hexokinase 1?

- In insulinsensitive tissues, e.g. skeletal muscle, in which hexokinase II is prominent, mitochondrial binding depends on the presence of insulin. - Hexokinase binds to porin, which forms a pore in the outer mitochondrial membrane through which metabolites pass. - Mitochondria were incubated with 32P labelled phosphate and unlabelled ATP. - The glucose-6-phosphate produced had a specific activity equal to that of the phosphate, indicating that ATP produced in oxidative phosphorylation was the source of the ATP used by hexokinase and not ATP in the medium. - This channelling of ATP is thought to be a means of coordinating the initial step of glucose metabolism with mitochondrial oxidative phosphorylation.

How does adrenaline effect heart cells?

- In normal heart muscle, Fru-2,6-P2 rises in response to adrenaline-mediated phosphorylation of 6PF-2-K/Fru-2,6-P2ase and accelerates glycolysis.

How does the structure of phosphatase change upon binding of AMP or phosphorylation?

- In phosphorylase a, the negatively charged phosphate on Ser-14 interacts with two positively charged Arg side chains, one on helix α2 of the same subunit and the other on the 'cap' of the other subunit. - This results in helix α2 and the cap being pulled together and the R state is stabilised. - The relative rotation of the two subunits in the T to R transition causes a tilt of the α7 helices, which are attached to segments of polypeptide that extend down into the active site more than 30 Å away. - This increases the binding of the negatively charged substrate Pi to positively charged Lys and Arg residues within the active site.

When can gradients of the metabolite form?

- In situations where there is a source and a sink for a metabolite.

What is the different effect PKA has in the liver compared to muscles and why is this.

- In the liver it favours gluconeogenesis and in the muscles it favours glycolysis. - The muscle isoforms of the bifunctional enzyme and pyruvate kinase PKA phosphorylation site.

How does a rise in glucose blood concentrations result in insulin release?

- Increased conc of glucose enters glut 2 transporters and is the substrate for glucokinase. - This increases flux through glycolysis and through the TCA cycle, leading to elevated ATP concentrations. - This results in the closure of ATP-sensitive K+ channels, plasma membrane depolarization and influx of Ca2+ through voltage gated Ca2+ channels, leading to insulin secretion. - The changes in plasma membrane [ATP] matched closely those of mitochondrial [ATP], suggesting that mitochondria located close to the plasma membrane may provide a localized pool of ATP. These mitochondria may also have privileged access to Ca2+, which activates the TCA cycle.

How is glucokinase controlled?

- Inhibited by a regulatory protein. - Fructose-6- phosphate, by binding to the regulatory protein, reinforces this inhibition and prevents a 'futile' cycle between glucose and glucose-6-phosphate. - The inhibition of glucokinase by the regulatory protein is antagonised by fructose-1- phosphate. - The stimulation of glucokinase by fructose-1- phosphate ensures that there is uptake by the liver of both glucose and fructose following a meal.

What does ATP metabolism in muscles depend on?

- Intensity of exercise - During prolonged exercise, glucose is oxidised aerobically to provide ATP. Because ATP is produced more slowly by oxidative phosphorylation than by anaerobic glycolysis, the intensity of exercise is necessarily reduced.

Describe the Cori cycle

- Lactate is broken down by lactate dehydrogenase into pyruvate. (ATP dependent) - Excess pyruvate converted to alanine.

What does lysine acetylation result in?

- Mass spectrometry analysis has demonstrated that virtually every enzyme in glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, the urea cycle, fatty acid metabolism, and glycogen metabolism is acetylated in human liver and that there are changes in the profile of acetylated metabolic enzymes with carbon source. Lysine acetylation uses acetyl-coenzyme A (CoA) as the acetyl group donor.

What are the two types of compartmentation?

- Metabolites may be compartmented as a result of sequestration within cellular organelles. - Compartmentation by binding, for instance ADP binding to actin in muscle cells.

What is the function of AMP-activated protein kinase?

- Nutrient or exercise-induced stress increases AMP levels and activates AMPK. - AMPK activation by AMP is allosteric and requires its phosphorylation on T172 by an upstream kinase (LKB1). - Binding of AMP leads to extrusion of a lipid anchor from AMPK, which tethers it to the plasma membrane close to - LKB1. Phosphorylation by LKB1 triggers AMPK activation. - The enzyme recognises ATP depletion and limits further utilisation by inhibiting glycogen, fatty acid and cholesterol synthesis. At the same time it initiates compensatory changes that maintain ATP levels e.g. by stimulating glucose transport and the β-oxidation of fatty acids.

How is HIF-1 regulated?

- Oxygen promotes the degradation of HIF-1α : prolyl-4-hydroxylase, which uses oxygen as a co-substrate, hydroxylates P564 and P402 of the α-subunit. This promotes interaction with the pVHL protein tumour suppressor protein which targets HIF-α ubiquitin dependent proteolysis. - Blocks its activity as a transcription factor: Hydroxylation of a conserved asparagine residue (N803) blocks interaction of a C terminal domain of the protein with p300/CBP transcriptional co-activators. - ARNT = HIF-1b

What are the allosteric effectors in muscle glycolysis

- PFK1 is allosterically inhibited by ATP (i.e. ATP is an allosteric effector as well as a substrate) and pyruvate kinase is allosterically activated by fructose-1,6-bisphosphate (called FDP in the diagram). - Inhibition of PFK1 by ATP is relieved by AMP. During exercise there is a large rise in [AMP]. - The increase in PFK1 activity results in an increase in the steady state concentration of fructose-1,6-bisphosphate, which is a positive allosteric effector of pyruvate kinase and a decrease in the concentration of glucose-6-phosphate, which is an inhibitor of hexokinase

How can you show that binding of HKII to heart mitochondria has functional consequences

- Perfusion of isolated hearts for 15 minutes with a cellpermeable peptide containing the HKII mitochondrial binding motif (TAT-HKII), which displaces HKII from the mitochondria, caused mitochondrial membrane depolarization, resulting in mitochondrial swelling and extensive structural disruption of the cardiac tissue. A control peptide(TAT-CON) was without effect.

Why do certain cancers produce a spliced version of Pyruvate kinase (PKM2).

- Phospho-tyrosine motifs, produced by growth factor signalling via protein tyrosine kinases, bind directly to PKM2, releasing its allosteric activator fructose1,6-bisphosphate. - The resulting inhibition of the enzyme leads to a build-up of upstream intermediates in the glycolytic pathway and diversion of glucose into lipid synthesis. - PKM2 is also involved in the cell's metabolic response to oxidative stress. - PKM2 is also involved in the cell's metabolic response to oxidative stress. Cys 358 in PKM2 is oxidized by hydrogen peroxide (H2O2), which decreases PKM2 activity, decreasing pyruvate formation and diverting flux from glycolysis into the pentose phosphate pathway (PPP). Increased levels of PEP inhibit triosephosphate isomerase and increase flux into the PPP

How does Phosphoglycerate mutase 1 control the flux into the pentose phosphate pathway?

- Phosphoglycerate mutase 1 (PGAM1), whose expression is negatively regulated by p53, regulates biosynthesis by controlling the levels of 3-phosphoglycerate (3-PG) and 2- phosphoglycerate (2-PG). - 3-PG binds to and inhibits 6-phosphogluconate dehydrogenase in the PPP, while 2-PG activates 3-phosphoglycerate dehydrogenase to provide feedback control of 3-PG levels by increasing flux into the pathway of serine biosynthesis.

What is the difference between the number of phosphorylation sites in Phosphorylase and glycogen synthase.

- Phosphorylase has one at ser-14 - Glycogen synthase has multiple phosphorylation sites (7), allowing its activity to be reduced more gradually.

What is the effect of phosphorylating the bifunctional 6PF-2-K/Fru-2,6-P2ase enzyme.

- Phosphorylation of the bifunctional enzyme 6PF-2-K/Fru-2,6-P2ase results in inhibition of its kinase and activation of its phosphatase activities. - The consequent fall in fructose-2,6-bisphosphate concentration, a potent allosteric activator of PFK1 and inhibitor of Fru-1,6-P2ase, leads to enhanced gluconeogenesis, inhibition of glycolysis and a decrease in the concentration of fructose 1,6- bisphosphate.

What is the general consensus sequences of kinases?

- Positively charged arginine and lysine sites towards the N terminal of the modified serine residual.

What do isotope labelling experiments shown about mitochondrial creatine kinase? What is the significance?

- Preferential access to ATP generated in the mitochondrial matrix. - It acts as a temporal energy buffer, maintaining ATP, ADP and H+ concentrations during periods of increased cellular activity; e.g. muscle contraction. - It maintains high local ATP/ADP ratios at sites of rapid ATP utilisation; e.g. the myofibrils. - It acts as an energy shuttle. Phosphocreatine generated at the mitochondria diffuses to the myofibrils, where it is used to phosphorylate ADP generated by the myofibrillar ATPase. Creatine diffuses back to the mitochondria and is used in another cycle. Net ATP transport from mitochondria to myofibrils occurs via phosphocreatine diffusion. Phosphocreatine and creatine are better suited as transport molecules than ATP or ADP, as they are present at higher concentrations. This is especially true for ADP, whose concentration is several orders of magnitude lower than that of creatine

How is Pyruvate kinase inhibited in the liver during gluconeogenesis?

- Pyruvate kinase is allosterically inhibited by ATP and alanine and activated by fructose1,6-bisphosphate. Phosphorylation (LIVER ISOFORM) also inhibits this enzyme by increasing its apparent KM for phosphoenolpyruvate, decreasing activation by fructose-1,6- bisphosphate and enhancing inhibition by ATP and alanine. - However, with saturating concentrations of fructose-1,6- bisphosphate, phosphorylation has no effect on activity

How does Protein phosphatase 1 (PP1) function and how is it regulated?

- Responsible for the removal of all of the phosphates involved in the regulation of glycogen metabolism, decreases the rate of glycogen breakdown and accelerates the rate of glycogen synthesis. - Phosphorylation of the G subunit by PKA prevents it from binding to the catalytic subunit of PP1. - Phosphorylation of the PP1 inhibitor results in its binding and inhibition of PP1. - Thus, as glycogen degradation is switched on by cAMP, the accompanying phosphorylation of inhibitor-1 and the G subunit inhibits PP1 and keeps phosphorylase in the active form and glycogen synthase in the inactive form.

- Why in brain tissue is the concentration of ADP in the cytosol very much less than the total concentration measured in tissue extracts? - How can this be overcome?

- Sequestration of ADP within the mitochondria. - The free cytosolic ADP concentration can be determined by measuring the tissue concentrations of the substrates of the enzyme creatine kinase i.e. creatine, creatine phosphate and ATP using NMR. -Keq is obtained using test tube experiments. - pH will give [H+]

What does phosphocreatine act as?

- Serves as a rapidly mobilizable reserve of high-energy phosphates in skeletal muscle, myocardium and the brain to recycle adenosine triphosphate, the energy currency of the cell. - Phosphocreatine is able to donate its phosphate group to convert ADP to ATP.

What is the advantages of protein phosphorylation

- Signal amplification: i.e. cAMP present at 1 μM is able to activate phosphorylase present at ~100 μM. - Co-ordination: between regulatory networks. Can upregulate one pathway and downregulate another. - Increased sensitivity: i.e. a small % change in initial signal produces a large % change in end product.

What happens to the liver during a fast such as overnight?

- Stimulates the breakdown of liver glycogen to release glucose into the bloodstream.

How does insulin function in metabolism?

- Stimulates the uptake of glucose by the muscles to get converted into muscle glycogen. - Also stimulates the uptake of glucose by the liver to be converted into glycogen.

What is substrate channelling?

- Substrate channeling is the passing of the intermediary metabolic product of one enzyme directly to another enzyme or active site without its release into solution (without fully equilibrating with the bulk phase).

How does a futile cycle lead to flux control?

- Substrate cycles provide a way of amplifying the effects of allosteric activators and inhibitors in order to increase the flux through the pathway.

What are the three different ways in which the output of a biological system can be controlled?

- Substrate level control. - Cooperativity - Ultrasensitivty by altering Vmax by coordinate control (molecular switch)

How can the 'shuttle' hypothesis be supported?

- The 'shuttle' hypothesis received strong support from studies on a mouse strain in which both alleles of the muscle creatine kinase gene had been disrupted (Cell (1993) 74, 621). The skeletal muscles of these animals adapted to loss of the enzyme by producing more and larger mitochondria, thus reducing the diffusion distance between mitochondria and myofibrils.

How does PEPCK get upregulated?

- The CREs (cAMP response elements), which are 8 bp palindromes, mediate the effect of cAMP on transcription. - A leucine zipper protein called CREB (cAMP response element binding protein) binds to this target DNA sequence. - Dimerisation of CREB, through its C-terminal leucine zipper domain, brings together two basic DNA binding modules that bind the palindromic CRE. - Phosphorylation of CREB by PKA promotes dimerisation and enhances transcriptional activation.

Describe the sequential model for co-operation.

- The binding of a ligand to one subunit changes the protein's shape, thereby making it more thermodynamically favourable for the other subunits to switch conformation to the high-affinity state. - Ligand binding may also result in negative cooperativity, or a reduced affinity for the ligand at the next binding site, a feature that makes the KNF model distinct from the MWC model, which suggests only positive cooperativity.

Why does Glucokinase display a sigmoidal saturation curve?

- The enzyme is thought to exist in two conformations that interconvert slowly. Open (low affinity) and closed (high affinity). - The low affinity form (E') predominates at low substrate concentrations and the high affinity form (E) at high substrate concentrations, giving rise to a sigmoidal saturation curve and enhanced sensitivity to glucose concentration

What is the the general trend in metabolism in cancer cells?

- The majority of human and animal tumours display a high rate of aerobic glycolysis. - The hypoxic environment of tumours selects for cells that are adapted to chronic hypoxia

How is phosphorylase kinase affected by Ca2+ and what is its relevance?

- The non-phosphorylated form can also be partly activated by Ca2+ binding to the δ subunit. - The δ subunit is calmodulin, a Ca2+ sensor that stimulates many enzymes in eukaryotes. - This mode of activation is important in muscle, where contraction is triggered by release of Ca2+ from the sarcoplasmic reticulum. - In liver, the release of Ca2+ produced by hormone activation of the phosphoinositide cascade is the most important route for phosphorylase kinase activation.

What membranes are freely permeable to glucose?

- The plasma membranes of β-cells and liver cells are freely permeable to glucose (they contain high levels of insulin-independent GLUT2 transporters).

Define flux

- The rate of turnover of molecules through a metabolic pathway.

What is the significance of there being almost 10 times as much phosphorylase a than PP1.

- There are ~10 phosphorylase a molecules per phosphatase, therefore the synthase activity will increase only when most of the phosphorylase a has been converted into the b form. - However, more recent work suggests that this system can be overridden by glucose-6- phosphate (G 6-P). Increases in G 6-P concentration promote the dephosphorylation and thus activation of glycogen synthase. - Shows a rapid control mechanism.

How does the phosphorylation of Glycogen synthase affect its function?

- This enzyme is phosphorylated on seven different serine residues by at least five different protein kinases. - Phosphorylation inhibits activity by increasing the KM for its substrate, UDPglucose, and the KD (dissociation constant) for its activator, glucose-6-phosphate. - The KDs for its inhibitors, ATP and Pi, are reduced; i.e. as with phosphorylase, the effect of phosphorylation is to change the response to allosteric effectors. The enzyme changes activity gradually as phosphorylation of all the different sites changes.

Why is AMP a good indicator of activity?

- This is a near-equilibrium reaction catalysed by adenylate kinase. - A 15% decrease in [ATP] results in a ~3x increase in [AMP]. AMP is a very sensitive indicator of cellular energy status. - The change in AMP concentration during exercise is much higher than for ADP.

At which amino acids does phosphorylation occur, and what is the net effect?

- Threonine, serine and tyrosine. - Gain of two negative charges.

How can PEPCH be regulated

- Under high concentrations of glucose PEPCK gets acetylated and therefore degregated by ubiquitination.

Describe the experiment used to prove that glucokinase is the sensor for glucose blood concentration in pancreatic B cells.

- Using transgenic mice expressing yeast hexokinase in β-cells which has a Km lower than normal blood glucose concentrations. - These mice were found to have increased insulin secretion at low blood glucose levels

What does a high protein concentration inside the cell result in?

- Will promote protein-protein interactions that are not observed in diluted cell extracts. These interactions may be further enhanced by solvent exclusion effects, which occur in solutions of macromolecules at high concentrations, and which can cause large increases in the activity coefficients of proteins.

What does the hill plot indicate about the function of cooperativity?

- cooperativity can make a system very sensitive to small changes in substrate concentration. - the value of n (known as the Hill coefficient) can act a useful empirical measure of cooperativity. As n increases, the sigmoidal curve becomes steeper or 'ultrasensitive' - n = number of binding sites assuming full cooperativity

Where does the hormonal control of liver glucose metabolism appears to convergeat?

- transcriptional co-activator PGC-1 - PGC-1 is induced in liver by fasting and results in the coordinate upregulation of PEPCK, glucose-6- phosphatase and fructose1,6-bisphosphatase gene expression.

What TF regulates glucose metabolism in the liver and how is it induced?

-A high carbohydrate diet induces ChRE-binding protein (ChREBP), a basic helixloop-helix leucine zipper transcription factor that binds to the carbohydrate response element (ChRE) in the L-type pyruvate kinase and GLUT2 promoter. - DNA binding is inhibited by phosphorylation by PKA and by AMPK

Where are the two different isotypes of creatine kinase located?

-I n skeletal muscle, the cytosolic form of the enzyme creatine kinase is associated with the myofibrillar M-band, where it is thought to be functionally coupled to the myofibrillar Mg2+ ATPase. - In addition, there is a mitochondrial isoform located on the outer surface of the inner mitochondrial membrane, which is thought to form a complex with the adenine nucleotide translocase in the inner membrane and porin in the outer membrane.

What is substrate cooperativity in relation to enzymes

-In enzymes or receptors that have multiple binding sites, the affinity of the binding sites for a ligand is apparently increased, positive cooperativity, or decreased, negative cooperativity, upon the binding of a ligand to a binding site

How is Phosphorylase activity controlled?

-The activity of this glycogen phosphorylase is subject to two control mechanisms, allosteric regulation and phosphorylation (controlled under adrenaline). - The less active b form is converted to the more active a form by phosphorylation at Ser-14 in each of the two subunits. - The enzyme is allosterically regulated by Pi and AMP (activators) and glucose-6-phosphate and ATP (inhibitors), the levels of which reflect the energy state of the muscle. - If the b form is already fully activated by AMP and Pi then phosphorylation has little further effect. - The regulation of phosphorylase can be viewed in terms of the concerted (MWC) allosteric model.

What is the net cost of gluconeogenesis?

2 ATPs (1 of which is GTP, an equivalent ATP)

How does AMP effect the rate of FBPase

Allosteric inhibitor

What technique can display the intracellular environment of the cell?

Cryogenic electron tomography

How is rate different from flux?

Describes the rate of a single enzyme in a pathway rather than in the whole metabolic pathway.

Where is FBPase absent and why?

FBPase is absent from muscles that are always mechanically active, such as heart muscle, and which do not have extreme variations in energy demand.

How does glucose gets trapped in a cell?

Gets phosphorylated.

What is the basic function of insulin?

Insulin stimulates glycogen synthesis by activating PP1. The enzyme phosphorylates the G subunit of PP1 at a different site from that modified by PKA and activates the enzyme. Consequent dephosphorylation of phosphorylase kinase, phosphorylase and glycogen synthase promotes glycogen synthesis and blocks degradation.

How is control through changes in enzyme concentration achieved in pathways involving glucose?

Insulin, which rises following feeding, stimulates the expression of PFK1, pyruvate kinase and 6PF-2-K/Fru-2,6-P2ase. Glucagon, which rises in starvation, inhibits the expression of these enzymes and stimulates production of phosphoenolpyruvate carboxykinase (PEPCK) and Fru-1,6-P2ase

What does the hill coefficient n tell you?

Is a measure of cooperativity in a binding process. A Hill coefficient of 1 indicates independent binding, a value of greater than 1 shows positive cooperativity binding of one ligand facilitates binding of subsequent ligands.

Define substrate level control

Modulating enzyme rate by controlling access to its substrate.

Other than the liver, where is glucokinase found?

Pancreatic beta-cells, functions as a sensor for blood glucose concentration.

What is generated during the amplification of glycolysis through the futile cycle?

This signal amplification is paid for by ATP hydrolysis, and the free energy released is dissipated as heat

Where does gluconeogenesis occur?

liver

What is the glucose sensor in liver cells?

phosphorylase a