Biochem Lec 23- Glycolysis Regulation and Gluconeogenesis

How many steps are considered irreversible in glycolysis (under cellular conditions)? What are they?

Three: Reactions 1,3, and 10

1. Reaction 1→ Hexokinase

• Glucose + ATP→ Glucose-6-phosphate (G-6P) + ADP + H⁺

2. Reaction 3→ Phosphofructokinase (PFK)

• Fructose-6-phosphate + ATP→ Fructose-1,6-bisphosphate (F-1,6-BP) + ADP + H⁺

3. Reaction 10→ Pyruvate Kinase

• Phosphoenolpyruvate→ Pyruvate

ALL have a negative free energy and are points of regulation

How does glycolysis regulation differ in muscle vs. liver?

Muscle:

• Rapid production of ATP during intense exercise/exertion

• Regulation is done by energy charge and the concentration of ATP vs. concentration of AMP

Liver:

• Glycolysis is used to provide carbon skeletons for biosynthesis

• Balance between gluconeogenesis and blood glucose during fasting

• Hormonal regulation is important

What is the key step of glycolysis regulation in muscle?

Phosphofructokinase (PFK):

• Committed step in glycolysis

• ATP: high energy→ - allosteric effector

1. ATP binds a regulatory site and reduces PFK’s affinity for Fructose-6-phosphate→ prevents wasteful use of glucose at rest

• AMP: Low energy→ + allosteric effector

1. During exercise, adenylate kinase makes AMP→ Rising AMP levels overrides ATP inhibition→ restores high affinity of PFK for F-6P and accelerates glycolysis to make ATP

Describe the structure of PFK. What is it and how does the concentration of ATP vs. AMP impact its activity (i.e., its kinetics)?

PFK is a classical allosteric enzyme:

• Tetramer

• Has 2 ATP binding sites

1. Active site→ high affinity

2. Allosteric binding site→ lower affinity

High [AMP] and low [ATP]→ increases reaction velocity at lower [Fructose-6-phosphate] and is hyperbolic in shape

Low [AMP] and high [ATP]→ decreases reaction velocity at lower [Fructose-6-phosphate] and is sigmoidal in shape

How do the concentrations of ATP and AMP compare in resting muscle? How does this impact glycolysis?

In resting muscle, [ATP] > [AMP]:

• PFK activity is inhibited→ ATP is a negative allosteric regulator of PFK

• Pyruvate kinase activity is inhibited→ ATP is also a negative allosteric regulator of pyruvate kinase

• PFK inhibition leads to accumulation of glucose-6-phosphate and feedback inhibits hexokinase:

1. Fructose-6-phosphate is not being converted by PFK→ increase in [F-6-P] shifts equilibrium of G-6-P→ F-6-P to favor the reactants and causes a build up of G-6-P

2. Build up of G-6-P inhibits hexokinase by shifting the equilibrium of Glucose→ G-6-P to favor the reactants and prevents further phosphorylation of glucose

High [ATP]/low [AMP] inhibits glycolysis at multiple steps (PFK, pyruvate kinase, and hexokinase indirectly) through allosteric and feedback inhibition→ glucose is not “burned”

How do the concentrations of ATP and AMP compare in working muscle? How does this impact glycolysis?

In working muscle [ATP] < [AMP]:

• PFK activity is stimulated→ AMP is pos. allosteric effector of PFK→ More fructose-1,6-bisphosphate is synthesized

• Pyruvate kinase is allosterically activated by fructose-1,6-bisphosphate (feed forward stimulation)→ ensures that as glycolysis speeds up, phosphoenolpyruvate is efficiently converted to pyruvate→ maintains high ATP output

• Because of PFK stimulation, glucose-6-phosphate moves through glycolysis and hexokinase is active

Low [ATP]/high [AMP] stimulates glycolysis at multiple steps→ glucose is “burned” to generate ATP for muscle contraction

Describe the importance of maintaining blood glucose.

• Human body requires 160 g of glucose per day for survival

• 120 g are required for brain function→ brain only uses glucose as fuel whereas most other tissues can use fats and other fuels

• During fasting (no consumption of carbohydrate fuels), we have resources (mostly from liver and muscle glycogen) for about 200 g of glucose

• During sustained starvation, muscle protein is sacrificed and amino acids are used to generate pyruvate→ converted to glucose in liver

• This process is referred to as gluconeogenesis

What poses a thermodynamic problem for gluconeogenesis?

Glycolysis is irreversible:

• Seven reactions of glycolysis are freely reversible

• Reactions catalyzed by Hexokinase (1), PFK (3), and Pyruvate Kinase (10) are not

• Four specific Gluconeogenesis enzymes are used to reverse these three steps

• This process is costly: 6 high energy phosphates are required to convert pyruvate to glucose

Reversing glycolysis as a means of generating glucose is energetically costly

What is gluconeogenesis? Briefly describe it.

Gluconeogenesis→ synthesis of glucose from non-carbohydrate precursors:

• Done primarily in liver

• Provides glucose for bloodstream during fasting

• Not exactly a reversal of glycolysis

• Four reactions are needed to bypass the three irreversible steps of glycolysis

• This pathway costs energy→ 6 high energy phosphates per glucose formed

What two enzymes are required to catalyze the conversion of pyruvate to phosphoenolpyruvate (reverse reaction 10)? 

Pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase

Describe the role of pyruvate carboxylase in the conversion of pyruvate to phosphoenolpyruvate.

• Catalyzes the carboxylation of pyruvate to oxaloacetate

• Requires biotin→ serves as a covalently attached prosthetic group

• This biotin carries an “activated CO₂”:

1. Biotin is covalently attached to a lysine on a flexible domain (BCCP→ biotin carboxyl carrier protein)

2. Long and flexible and moves between two catalytic domains in pyruvate carboxylase

3. Biotin carboxylase (BC)

Biotin + ATP + HCO₃^- → Carboxybiotin + ADP + P_i

4. Carboxyl transferase (CT)

Carboxybiotin + pyruvate→ Biotin + Oxaloacetate

• This step REQUIRES ATP

Pyruvate + CO₂ + ATP + H₂O→ Oxaloacetate + ADP + P_i + 2H⁺

Describe the structure of pyruvate carboxylase and how it relates to biotin.

• Pyruvate carboxylase is a complex enzyme (1154 amino acids) that has multiple domains

• The two enzymatic domains are the biotin carboxylase (BC) and the carboxyl transferase (CT) domains→ the each have their own active site

• The biotin carboxyl carrier domain (BCCP) has a biotin co-enzyme attached to a lysine residue

• BCCP and biotin swing between the BC and CT active sites

• PT is the protein tetramerization domain which is not part of catalysis

Describe the role of phosphoenolpyruvate carboxykinase in the conversion of pyruvate to phosphoenolpyruvate.

• Oxaloacetate is decarboxylated and phosphorylated to generate phosphoenolpyruvate

• Carboxylation/decarboxylation is a way around having to phosphorylate pyruvate directly

Oxaloacetate + GTP→ Phosphoenolpyruvate + GDP + CO₂

What is the sum of “bypass” reactions?

Pyruvate + ATP + GTP + H₂O→ Phosphoenolpyruvate + ADP + GDP + P_i + 2H⁺

Only “irreversible” steps need to be bypassed by new steps. Why is this?

Non-irreversible steps are near equilibrium under cellular conditions, so the pathway can readily run in reverse

What enzyme reverses the third reaction in glycolysis (catalyzed by PFK)?

Fructose-1,6-bisphosphatase:

Fructose-1,6-bisphosphate + H₂O→ Fructose-6-phosphate + P_i

• Removes a phosphate from the 1 position

What enzyme reverses the first reaction in glycolysis (catalyzed by hexokinase)?

Glucose-6-phosphatase:

Glucose-6-phosphate + H₂O→ Glucose + P_i

• Removes a phosphate from the 6 position

• In most tissues, free glucose is not generated and glucose-6-phosphate (which cannot be transported out of the cell) is processed in some other way→ e.g., to form glycogen

Explain gluconeogenesis vs. “reverse glycolysis”

• In gluconeogenesis, 6 high-transfer-potential phosphoryl groups are spent to synthesize glucose from pyruvate:

1. 4 ATP

2. 2 GTP

• 2 are gained through glycolysis (2 ATP)→ the 4 extra (2 ATP and 2 GTP) are required to turn an unfavorable reaction into a favorable one

Reverse glycolysis:

2 Pyruvate + 2 ATP + NADH + 2 H₂O→ Glucose + 2 ADP + 2 P_i + 2 NAD⁺ +2H⁺

ΔGo’= +90 kJ/mol

Gluconeogenesis:

2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 6 H₂O→ Glucose + 4 ADP + 2 GDP +   6 P_i + 2 NAD⁺ + 2H⁺

ΔGo’= -48 kJ/mol

Recap glycolysis and gluconeogenesis.

• The reactions and enzymes involved differ at a few specific points

• These points are the “irreversible” steps of glycolysis (have large negative ΔGo’) that require specific enzymes to bypass

• This requires energy (4 ATP and 2 GTP per glucose)

• Key “irreversible” steps are control points for regulation

Image:

Red→ distinctive reactions that differ from glycolysis. The enzymes are located in the cytosol except for pyruvate carboxylase which is in the mitochondria