Lecture 3 Coordinated Regulation of glycolysis and gluconeogenesis

Question 1

What kind of pathway is glycolysis?

Answer 1

oxidative pathway

Question 2

What two reductive processes sustain glycolysis?

Answer 2

• Fermentation → In the absence of oxygen, fermentation provides glycolysis with the NAD+ necessary and gets rid of the electrons in NADH produced through glycolysis, as pyruvate is reduced to lactate or ethanol.
• Gluconeogenesis → When glucose is needed, gluconeogenesis takes place and provides NAD+ that will be necessary for the subsequent/eventual oxidation of glucose (as gluconeogenesis uses NADH as reducing power).

Question 3

What oxidative process sustains glycolysis?

Answer 3

ETC → In the presence of oxygen, the mitochondrial electron transfer chain oxidizes NADH to yield NAD+, while generating a proton and electric potential gradient that enables ADP phosphorylation to ATP.

Question 4

What 4 mechanisms affect flux through biochemical pathways?

Answer 4

• Organ-specific expression of hexokinases
• Allosteric control
• Covalent modification
• Substrate Cycles
• Genetic Control

Question 5

Allosteric control

Answer 5

Inhibition or activation of an enzyme by a small regulatory molecule (effector/ modulator) that interacts at a site (allosteric site) other than the active site (at which catalytic activity occurs).

Question 6

What are common effects of allosteric regulation?

Answer 6

Effectors are often substrates or products within the pathway. Allosteric effectors of enzymes catalyzing irreversible reactions have a rapid and dramatic impact on the flux through the pathway.

Question 7

What 3 enzymes of glycolysis are known to be allosteric?

Answer 7

• phosphofructokinase (reaction 3)
• glyceraldehyde-3 phosphate dehydrogenase (reaction 6)
• pyruvate kinase (reaction 10)

Question 8

What shape do allosteric enzymes have?

Answer 8

sigmoidal shape

Question 9

What are the 2 mechanisms for allosteric control of hexokinase?

Answer 9

• Mechanism 1 (DIRECT) → Hexokinase is allosterically inhibited by G6P (end-product negative feedback).
• Mechanism 2 (INDIRECT) → When PFK-1 activity is low, [F6P] increases and, consequently, [G6P] increases. G6P causes allosteric inhibition of hexokinase (by mechanism 1).

Question 10

What are the effectors of PFK-1?

Answer 10

reaction 3

• ATP inhibits
• AMP, ADP stimulates
• citrate inhibits
• Fructose 2,6-bisphosphate activates

Question 11

Regulation of PFK by ATP and AMP/ADP

Answer 11

• Low ATP but high AMP/ADP → shifts to the left so PFK-1 has greater affinity for F6P
• High ATP but low AMP/ADP → shifts to the right so PFK-1 has reduced affinity for F6P

Question 12

Role of citrate in allosteric control of PFK-1

Answer 12

Citrate forms downstream in another pathway when there is a lot of pyruvate and its accumulation can inhibit PFK-1

Question 13

How does fructose affect citrate?

Answer 13

Fructose will feed into glyocolysis and can become citrate downstream. Citrate does not inhibit the PFK1 the same way here and citrate can accumulate since fructose happens downstream of PFK1 and so you form ATP but the accumulated citrate can leave the mitochondria and become fat.

Question 14

What is F2,6BP?

Answer 14

The sugar called fructose-2,6-bisphosphate (F2,6BP) is made by a bifunctional enzyme called “PFK-2”, which phosphorylates F6P at carbon 2.

• PFK-2 is a bifunctional enzyme. The N-terminal domain has PFK-2 activity while the C-terminal domain has FBPase-2 activity.
• F2,6BP is not an intermediate in glycolysis.
• F2,6BP is the most important allosteric activator of PFK-1.
• In addition, F2,6BP inhibits FBPase-1 and, consequently, gluconeogenesis

Question 15

Role of F2,6BP in glycolysis and gluconeogenesis

Answer 15

Glycolysis: major allosteric activator

• PFK-1 is virtually inactive without F2,6BP
• F2,6BP binding to PFK-1 increases PFK-1 affinity for F6P (substrate) thus enabling glycolysis
• F2,6BP decreases PFK-1 affinity for ATP and citrate (which are allosteric inhibitors).

Gluconeogenesis:

• Conversely, F2,6BP binding to fructose bisphophatase (reverse/bypass reaction 9 of gluconeogenesis), allosterically decreases the FBPase affinity for F1,6BP (substrate) thus F2,6BP prevents gluconeogenesis

Question 16

Substrate cycle

Answer 16

A substrate cycle is a set of metabolic reactions, arranged in a loop, which does not result in net consumption or production of the metabolites.

• Often also labeled as futile cycles for the seemingly wasteful energy expenditure, substrate cycles have physiological functions, for example, in thermogenesis.

Question 17

Where does substrate cycling occur in glycolysis?

Answer 17

Reaction 3

• PFK-1 of glycolysis and FBPase-1 of gluconeogenesis form a substrate cycle.
• When the rates of PFK-1 and FBPase-1 are equal, the flux through the pathway is 0. This is a futile cycle. Bees use this substrate cycle to generate heat.

Question 18

Regulation of pyruvate kinase

Answer 18

Reaction 10

• Positive feedback (not allosteric, but thermodynamic ↔︎ ∆G)
  
  • PFK-1 activity ‘pushes’ pathway forward through increasing [F2,6BP], triose phosphates…[PEP]
• Negative feedback (allosteric)
  
  • End-products (alanine, ATP, Acetyl-CoA) inhibit pyruvate kinase allosterically

Question 19

What is a sensitive indicator of energy status?

Answer 19

The ratio of AMP/ATP. Moreso than the ADP/ATP ratio.

• Muscle contraction hydrolyzes ATP → ADP + Pi.
• Interestingly, metabolism somehow maintains [ATP] much higher than [AMP].
• Moreover, changes in [AMP] are dramatic (e.g., 600% increase following muscle contraction as per Table) while [ADP] is nearly unchanged due to its interconversion to both ATP and AMP by adenylate kinase (2 ADP → ATP + AMP) => a change in cellular [AMP] indicates a change in cellular energy status.