13 - Glycolysis regulation


  • Glycolysis produces pyruvate level by substrate level phosphorylation 


Isoenzymes - allow the function of an enzyme to work under various conditions, at different stages or in a variety of cellular conditions such as cancer


  • Catalyse the same reaction but have different regulation 

  • Right enzyme can be made at the right time 




Adapt depending on:

  • Location 

  • Situation 

  • Cellular condition 








3 irreversible enzymes of glycolysis: 

  1. Hexokinase 

  2. Phosphofructokinase - regulation at step 3 

  3. Pyruvate kinase - regulation at step 10 


  • Very negative delta G so reaction is irreversible 


Allosteric controls = milliseconds

Regulation by phosphorylation = seconds 

Transcriptional control = hours


 Role of glycolytic pathway

  • Degrades glucose to generate ATP 

  • Provides building blocks for biosynthetic reactions 









Glycolysis in muscles 

 

Reaction 1: Phosphorylation of glucose by hexokinase provides cellular advantages 



  • Incorporation of a phosphate into glucose in this energetically favoured reaction is important:

  • Glucose is a neutral molecule so it can diffuse across the membrane BUT phosphorylation confers a negative charge on glucose 

  • The membrane is essentially impermeable to glucose-6-phosphate 

  • Rapid conversion of glucose to glucose-6-phosphate keeps the intracellular concentration of glucose low, favouring diffusion of glucose into the cell 



  • Hexokinase is inhibited by glucose - 6 - phosphate

  • Reaction is essentially irreversible (commits the cell to taking up glucose) since coupled with ATP hydrolysis 

  • ATP is complexed with Mg2+ and its hydrolysis drives the reaction 




Reaction 3: ATP drives the phosphorylation of fructose-6-phosphate in a committed step

  • Commits the cell to glycolysis - rather than converting it to another sugar or storing it 

  • Large net decrease in free energy - serves as a point of regulation- because it contributes so much to the overall pathway

  • Prevents any later product (trioses) from diffusing out of the cell (both will have a phosphate group attached) 



Phosphofructokinase separates its catalytic and allosteric sites 


  • E. coli enzyme comprises of a tetramer of four identical subunits 

  • Each subunit of the human liver enzyme consists of 2 domains that are similar to the E. coli enzyme 


Regulation of PFK-1 

  • Dictated by energy needs of cell 

  • Inhibited by ATP 

  • ATP concentration does not vary greatly 

  • Only 10% change in [ATP] for resting vs vigorous exercise 

  • However, the rate of glycolysis varies more - as it relies on the [AMP] signal generated by adenylyl kinase

  • There are 2 sites on the enzyme for ATP binding

  • High affinity site (activity) 

  • Low affinity inhibitory site (regulatory / allosteric site) 



  • Small reductions in ATP lead to larger increases in AMP helping turn on PFK 


  • Adenylyl kinase rapidly interconverts ADP, ATP and AMP to maintain this equilibrium

  • When ATP needs are high - adenylyl kinase generates ATP from 2ADP 

  • An 8% hydrolysis of ATP = two-fold increase in ADP and four-fold increase in AMP






Reaction 10; Pyruvate kinase makes pyruvate 


  • Pyruvate kinase mediates the transfer of a phosphoryl  from phosphoenolpyruvate to ADP to make ATP and pyruvate 



  • Pyruvate is more stable on its own 


  • The large negative delta G of this reaction makes pyruvate kinase a suitable target site for regulation of glycolysis 

  • This is a coupled reaction allowing the formation of ATP 

  • Reaction requires Mg2+ ion and is stimulated by K+ 

  • Pyruvate kinase reaction equilibrium lies very far to the right 











Regulation of pyruvate kinase 

  • It possesses allosteric sites for numerous effectors 

  • Activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl CoA




Summary of glycolysis in muscles 

  • Hexokinase is allosterically inhibited by glucose - 6 - phosphate 

  • Pyruvate kinase is inhibited by the allosteric signals ATP and alanine, and stimulated by fructose-1,6-bisphosphate (the product of the phosphofructokinase reaction) 

  • In muscle, glycolysis is regulated to meet the energy needs of contraction 




At rest, glycolysis not very active 


Glucose-6-phosphate converter into glycogen 

















Regulation of glycolysis in the liver 


  • More diverse biochemically than muscle 

  • Liver maintains blood-glucose levels: stores glucose as glycogen when glucose is plentiful 

  • Release of glucose when supplies are low 

  • Use of glucose for generation of reducing power for biosynthesis & to synthesise biochemicals 

  • Generate TCA intermediates and acetyl CoA to synthesise fats 



  • Hexokinases 1, 2, 3 have high affinities for glucose with Km’s around 0.1mM 

  • They’re inhibited by G-6-P - the product of the reaction 

  • This allows the brain and muscles to process glucose when there is a low concentration and not waste if not needed

  • Hexokinase 4 or glucokinase - not only found in liver - catalyses the same reaction but has a Km of ~10mM - and not inhibited by G-6-P

  • When glucose levels are high - these properties enable the liver to produce G-6-P for conversion to G-1-P for glycogen production as well as to pyruvate for fat production



Km values 

  • Similar to Kd values 

  • They state the binding affinity of the substrate to the enzyme

  • Lower value = tighter binding [can bind to substrate even if substrate levels are low] 

  • Higher value = looser binding  



Isoenzymes in the muscles vs. liver 



Reasons for differences in hexokinase usage: 

  • Muscle - geared to breaking down glucose when it requires more energy

  • Liver - geared to break down glucose to store excess glucose as glycogen or as fats by degrading it to acetyl CoA 



Insulin usage 

  • Hormone that affects metabolism and body systems 

  • Causes body cells to take up glucose from blood - to be stored as glycogen in liver and muscle 

  • Stops the use of fat as an every source that is mobilised in the liver 

  • When insulin is absent (or low) glucose is taken up and liver begins to use fat as energy source 

  • When insulin control fails - diabetes mellitus 



Insulin vs glucagon 


BG levels 


Blood glucose usually maintained between 70 mg/dl (3.9mM) and 110 mg/dl (6.1mM)


<70 mg/dl = hypoglycemia 


>180 mg/dl = hyperglycemia 












Insulin 

  • Insulin secreted from pancreatic islet beta cells 

  • Response to high blood sugar 


Glucagon 

  • Secreted by pancreatic alpha cells 

  • Response to low BG levels (between meals and during exercise) 

  • Greatest effect on the liver 



Glucokinase as a glucose sensor and drug target for diabetes 

  • Due to large conformational change on glucose binding 

  • In islet beta cells - glucokinase ‘senses’ glucose levels (via change into active conformation) - leading to insulin response - ideal for this role due to being sensitive to small changes 



  • People with low GK activity have impaired insulin response - may acquire diabetes 

  • Small molecule synthetic activators have been used in murine models that can activate GK and decrease BG levels 



  • Active form of GK can transmit signals via transduction pathway - leads to release of insulin 


Phosphofructokinase 


Key liver specific regulators are: 

  • Inhibitor citrate (reports on the status of the citric acid cycle) 

  • Activator fructose 2,6 bisphosphate (helps liver respond to blood glucose levels) 

In the liver, phosphofructokinase 2 (PFK2) generates fructose 2,6-bisphosphate




Effect of F-2,6-BP on PFK1

  • It is an allosteric inhibitor










Effect of F-2,6-BP on ATP saturation curve 

  • It decreases the inhibition of ATP 



Feedforward stimulation 

  • Fructose-2,6-bisphosphate is an allosteric activator of PFK1 and stops inhibition by ATP

  • Occurs in the liver 






PFK energy regulation in most cells 

  • F-6-P, AMP stimulate glycolysis 

  • High ATP decreases glycolysis 

  • PFK is inhibited by citrate 

  • High level of citrate mean that biosynthetic precursors are abundant and so additional glucose should not be degraded for this purpose 





PFK regulation in liver cells 

  • Allows feedforward stimulation to lower BG levels 

  • In the liver, high BG stimulates PFK2 which makes F-2,6-BP which activates PFK1 - this will metabolise the glucose 





Pyruvate kinase

  • Regulated by allosteric effectors and covalent modification 

  • Tetramer of 57-kDa subunits


 

  • Liver pyruvate kinase (PK) can also control BG levels


Isoenzyme forms: 

  • Both have many common properties 

L = predominates in liver 

  • Catalytic properties controlled by reversible phosphorylation 

M = predominates in the brain



Immediate energy needs vs. maintaining glucose levels for all tissues (liver only) 

Step 1: 

  • Muscle/liver hexokinase vs. liver glucokinase

  • GK is active only after a meal, when BG levels are high & turns on insulin production 


Step 2: 

  • Muscle PFK vs. Liver PFK (additional role of F-2,6-BP to respond to high glucose levels with insulin) 


Step 10: 

  • Muscle PK vs. Liver PK (additional role of glucagon to respond to low glucose) 

  • Energy need response is quite local

  • Glucose level response mediated by hormones (but in the case of insulin sensed locally by GK) 

  • Enzymesare tuned to different signals in order to best serve their particular cell/tissue 






Insulin signals the fed state and tells the liver and muscle to process / store glucose to maintain BG levels