Problem 3 - Biochemistry: A Short Course

Question

What reactions of glycolysis are not reversible under intracellular conditions? How are these reactions bypassed in gluconeogenesis?

Step-by-Step Solution

Answer

The irreversible steps in glycolysis are bypassed in gluconeogenesis through specific enzymes: glucose-6-phosphatase, fructose-1,6-bisphosphatase, and pyruvate carboxylase with PEPCK.

Step 1: Identify Irreversible Reactions in Glycolysis

Glycolysis has three key irreversible reactions. These occur at the following steps: (1) conversion of glucose to glucose-6-phosphate by hexokinase/glucokinase, (2) conversion of fructose-6-phosphate to fructose-1,6-bisphosphate by phosphofructokinase-1 (PFK1), and (3) conversion of phosphoenolpyruvate (PEP) to pyruvate by pyruvate kinase. These reactions are irreversible due to their large negative Gibbs free energy changes under physiological conditions.

Step 2: Bypass One - Glucose to Glucose-6-Phosphate

In gluconeogenesis, the conversion of glucose-6-phosphate back to glucose does not use hexokinase/glucokinase. Instead, it uses the enzyme glucose-6-phosphatase, which removes the phosphate group from glucose-6-phosphate, allowing free glucose to be formed and released into the bloodstream.

Step 3: Bypass Two - Fructose-1,6-Bisphosphate Conversion

To bypass the fructose-6-phosphate to fructose-1,6-bisphosphate conversion, gluconeogenesis uses the enzyme fructose-1,6-bisphosphatase. This enzyme catalyzes the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate, bypassing the irreversible phosphofructokinase-1 step.

Step 4: Bypass Three - Pyruvate to Phosphoenolpyruvate (PEP)

The conversion of pyruvate to PEP is bypassed in gluconeogenesis by a two-step reaction. First, pyruvate is converted to oxaloacetate by pyruvate carboxylase. Then, oxaloacetate gets converted to PEP by the enzyme phosphoenolpyruvate carboxykinase (PEPCK). These steps bypass the irreversible conversion catalyzed by pyruvate kinase in glycolysis.

Key Concepts

Irreversible Reactions in Glycolysis

In the pathway of glycolysis, certain reactions proceed in one direction under cellular conditions, meaning they are irreversible. This primarily occurs due to a significant decrease in Gibbs free energy during these reactions. In glycolysis, three key reactions are considered irreversible. These reactions happen at specific steps:

• The conversion of glucose to glucose-6-phosphate.
• The transformation of fructose-6-phosphate into fructose-1,6-bisphosphate.
• The conversion of phosphoenolpyruvate (PEP) to pyruvate.

To understand why these reactions are irreversible, it's important to recognize that the large negative change in Gibbs free energy makes it energetically unfavorable for these reactions to proceed in the reverse direction. In cellular metabolism, such reactions set the directionality of metabolic pathways, ensuring a constant flow towards the end products. During gluconeogenesis, these irreversible steps are bypassed by alternative enzymes to facilitate the synthesis of glucose, reversing glycolysis.

Hexokinase and Glucokinase

The first step in glycolysis involves a crucial phosphorylation reaction, where glucose is converted into glucose-6-phosphate. This reaction is driven by the enzymes hexokinase and glucokinase. Both enzymes catalyze the transfer of a phosphate group from ATP to glucose, converting it into a form that remains trapped inside the cell.

• **Hexokinase** operates in most tissues and has a high affinity for glucose, allowing it to function effectively even at low glucose concentrations.
• **Glucokinase** is found in the liver and pancreas, with a lower affinity, allowing it to act when glucose levels are high, such as after a meal. This characteristic helps in regulating blood sugar levels.

In gluconeogenesis, the bypass of this irreversible step is accomplished by the enzyme glucose-6-phosphatase, which removes the phosphate from glucose-6-phosphate, releasing free glucose into the bloodstream. This enzyme is crucial for maintaining blood glucose levels, especially during fasting.

Phosphofructokinase-1

Phosphofructokinase-1 (PFK-1) is a key regulatory enzyme in glycolysis, responsible for the irreversible phosphorylation of fructose-6-phosphate into fructose-1,6-bisphosphate. This step is often regarded as the committed and most regulated step of glycolysis. PFK-1 controls the pace of the glycolytic pathway and responds to various allosteric regulators, ensuring energy demand is adequately met.

• **AMP and fructose-2,6-bisphosphate** act as activators of PFK-1, signaling the need for more ATP or energy in the cell.
• **ATP and citrate** inhibit PFK-1, indicating sufficient energy levels or a high rate of fatty acid or amino acid synthesis.

A hallmark of gluconeogenesis is the bypassing of this step through the enzyme fructose-1,6-bisphosphatase, which converts fructose-1,6-bisphosphate back into fructose-6-phosphate. By doing this, gluconeogenesis ensures a steady supply of glucose, especially during periods of fasting or intense exercise.

Pyruvate Kinase

Pyruvate kinase plays an essential role in the final step of glycolysis, where it catalyzes the transfer of a phosphate group from PEP to ADP, yielding ATP and pyruvate. This reaction also illustrates an irreversible step in glycolysis, crucial for energy generation in cells.

• Pyruvate kinase activity is regulated by various factors such as phosphorylation and allosteric effectors to fine-tune the metabolic needs of cells.
• The availability of pyruvate allows cells to further metabolize it either aerobically, in the mitochondria, or anaerobically, in the cytoplasm.

In gluconeogenesis, the reversal of the reaction carried out by pyruvate kinase is achieved through a two-step process: the conversion of pyruvate to oxaloacetate by pyruvate carboxylase, and subsequently, the conversion of oxaloacetate to PEP by phosphoenolpyruvate carboxykinase (PEPCK). These steps are pivotal in enabling the synthesis of glucose from non-carbohydrate sources.