Glycolysis Notes

Glycolysis

Glycolysis is a catabolic process that degrades glucose (and other simple sugars) in nearly every living cell. It occurs in the cytosol of cells and is an anaerobic process.

18.1 Essential Features of Glycolysis

Glycolysis, also known as the Embden-Meyerhof pathway, converts one molecule of glucose into two molecules of 3-carbon pyruvate through 10 enzyme-catalyzed steps. In brain, kidney medulla, rapidly contracting skeletal muscles, erythrocytes, and sperm cells, glucose is the primary source of metabolic energy. Pyruvate is a versatile metabolite with several potential fates.

Pyruvate Fates

Under aerobic conditions, pyruvate is converted to Acetyl-CoA, which enters the TCA cycle, ultimately producing $4CO<em>2$ and $4H</em>2O$. Under anaerobic conditions, pyruvate can be converted to lactate (in animals and plants, and contracting muscle) or ethanol + $CO_2$ (in yeast). Both anaerobic processes regenerate $NAD^+$.

Glycolysis Phases

Glycolysis consists of two phases:

• Phase 1: Glucose is broken down into two molecules of glyceraldehyde-3-phosphate (G-3-P). This phase requires an investment of 2 ATP molecules.
• Phase 2: Two molecules of G-3-P are converted into two molecules of pyruvate, producing 4 ATP and 2 NADH molecules.

18.2 Coupled Reactions

Glycolysis converts some of the metabolic energy of glucose into ATP. While not all energy is converted, the production of two ATP molecules is an energy-requiring process. Free energy from coupled reactions drives the synthesis of ATP.

Thermodynamics

For a reaction $A + B \rightleftharpoons C + D$, the equilibrium constant $K_{eq} = \frac{[C][D]}{[A][B]}$.

The standard free energy change is denoted by $\Delta G°'$, and the free energy change $\Delta G$ is given by:

$\Delta G = \Delta G°' + RT\ln K_{eq}$, where R is the gas constant and T is the absolute temperature.

At equilibrium, $\Delta G = 0$, so $\Delta G°' = -RT\ln K_{eq}$.

• If $\Delta G°' > 0$, then $K_{eq} < 1$ (non-spontaneous).
• If $\Delta G°' = 0$, then $K_{eq} = 1$ (equilibrium).
• If $\Delta G°' < 0$, then $K_{eq} > 1$ (spontaneous).

18.3 Phase 1 Chemical Principles

The first phase of glycolysis involves phosphorylation and cleavage to reorganize glucose. Glucose is phosphorylated at C-1 and C-6, and the six-carbon skeleton is cleaved into two three-carbon molecules of glyceraldehyde-3-phosphate (G-3-P).

Reaction 1: Phosphorylation of Glucose

Glucose is phosphorylated at C-6 to form glucose 6-phosphate (G-6-P) by hexokinase or glucokinase. This is a priming reaction that requires ATP to provide energy and a phosphoryl group.

ATP hydrolysis makes the phosphorylation of glucose spontaneous. Under standard-state conditions (1M, 25°C), ATP hydrolysis releases 30.5 kJ/mol, while glucose phosphorylation requires 13.8 kJ/mol. Under cellular conditions, the $\Delta G$ of reaction 1 is -33.9 kJ/mol.

Cellular Advantages of Phosphorylating Glucose

• Phosphorylation keeps the substrate within the cell because glucose can diffuse across the cell membrane, but negatively charged glucose-6-phosphate cannot.
• Conversion facilitates the diffusion of glucose into the cell because conversion of glucose to glucose-6-phosphate maintains a low intracellular glucose concentration.

Isozymes of Hexokinase

Hexokinase requires $Mg^{2+}$ and has four isozymes:

• Hexokinase I-III: Hexokinase I is the primary form in the brain, while skeletal muscle contains a mixture of types I and II. These are allosterically inhibited by G-6-P.
  • Low $K_m$: high affinity for glucose
  • Low $V_{max}$: limited capacity to process glucose
  • Inhibited by G-6-P
• Hexokinase IV (Glucokinase): Found mainly in the liver and pancreas and functions only when glucose concentrations are high, effectively removing glucose from the blood. Controlled by insulin and relatively unaffected by G-6-P. It has a high $K_m$.
  • High $K_m$: requires high glucose concentration for half-saturation
  • High $V_{max}$: effectively removes glucose from the blood
  • Relatively unaffected by G-6-P
  • Controlled by insulin

Metabolic Fates of Glucose-6-Phosphate

Glucose-6-phosphate can be used in several pathways, including glycogen synthesis, the pentose phosphate pathway (synthesis of NADPH and 4-C, 5-C, and 7-C sugars), and glycolysis.

Reaction 2: Phosphoglucoisomerase

Phosphoglucoisomerase catalyzes the isomerization of glucose-6-phosphate to fructose 6-phosphate (F-6-P). This involves shifting the carbonyl oxygen from C-1 to C-2, converting an aldose into a ketose. The hemiacetal -OH of glucose is more difficult to phosphorylate than a simple primary hydroxyl and this isomerization activates C-3 for C-C bond cleavage in the fourth step of glycolysis.

Phosphoglucoisomerase, also known as phosphoglucose isomerase or glucose phosphate isomerase, requires $Mg^{2+}$ for activity and is highly specific for G-6-P. The $\Delta G°'$ is 1.67 kJ/mol, and the value of $\Delta G$ under cellular conditions is -2.92 kJ/mol (readily reversible).

This reaction proceeds through an enediol intermediate.

Reaction 3: Phosphofructokinase

ATP drives a second phosphorylation by phosphofructokinase, phosphorylating the carbonyl oxygen on C-1 to form fructose-1,6-bisphosphate (FBP). This second priming reaction requires ATP hydrolysis, making the phosphorylation of F-6-P spontaneous. The $\Delta G°'$ of phosphorylation of F-6-P is 16.3 kJ/mol, but when coupled with ATP hydrolysis, the $\Delta G°'$ is -14.2 kJ/mol ($\Delta G$ is -18.8 kJ/mol in erythrocytes.)

Regulation of Phosphofructokinase (PFK)

• PFK is the “valve” controlling the rate of glycolysis. It is allosterically regulated and utilizes ATP as both a substrate and an inhibitor. Citrate is an allosteric inhibitor of PFK; when the citric acid cycle reaches saturation, glycolysis slows down.
• PFK has two distinct binding sites for ATP: a high-affinity substrate site and a low-affinity regulatory site.
• High ATP concentrations cooperatively increase the $K_m$ for F-6-P.
• Low ATP concentrations: AMP reverses the inhibition due to ATP. Adenylate kinase interconverts ADP, ATP, and AMP.
• β-D-fructose-2,6-bisphosphate (F2,6-BP) regulates PFK. F2,6-BP increases the affinity of PFK for the substrate F-6-P and decreases the inhibition of PFK due to ATP.

PFK Activity:

• Increased when energy status falls (high [AMP])
• Decreased when energy status is high (high [ATP], high [citrate])
• F2,6-BP upregulates PFK.

Reaction 4: Fructose Bisphosphate Aldolase

Cleavage by fructose bisphosphate aldolase cleaves FBP between C-3 and C-4, yielding two triose phosphates: dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G-3-P). At physiological concentrations, the reaction is essentially at equilibrium. $\Delta G°'$ of cleavage is +23.9 kJ/mol, but $\Delta G$ in erythrocytes is -0.23 kJ/mol.

There are two classes of aldolase enzymes:

• Class I: Found in animal tissues, these aldolases form a covalent Schiff base intermediate between an active-site lysine and the carbonyl group of the substrate. They do not require a divalent metal ion.
• Class II: Found in bacteria and fungi, these aldolases do not form a covalent E-S intermediate and require a divalent metal ion (typically $Zn^{2+}$).

Reaction 5: Triose Phosphate Isomerase

Triose phosphate isomerase converts DHAP to G-3-P, making the C-1, C-2, and C-3 of glucose equivalent to the C-6, C-5, and C-4 positions. This reaction proceeds through an enediol intermediate.

18.4 Phase 2 Chemical Principles

The second phase of glycolysis converts the metabolic energy in the glucose molecule into ATP. Four new ATP molecules are produced through phosphoryl transfer from 1,3-BPG to ADP and then phosphoryl transfer from PEP to ADP.

Reaction 6: Glyceraldehyde-3-Phosphate Dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) oxidizes the aldehyde group of G-3-P to form 1,3-bisphosphoglycerate (1,3-BPG), a high-energy phosphate compound. This reaction also reduces $NAD^+$ to NADH.

Arsenate can act as a substrate in the G3PDH reaction, forming 1-arseno-3-phosphoglycerate, which breaks down to 3-phosphoglycerate. This bypasses ATP formation in glycolysis.

Reaction 7: Phosphoglycerate Kinase

Phosphoglycerate kinase (PGK) transfers a phosphoryl group from BPG to ADP, forming 3-Phosphoglycerate (3-PG) and ATP. $Mg^{2+}$ is required for PGK activity. This is referred to as substrate-level phosphorylation, where ADP is phosphorylated to form ATP at the expense of a substrate (G-3-P).

2,3-BPG is synthesized and metabolized by a pair of reactions that make a detour around the PGK reaction involving phosphoryl transfer from the C-1 position of 1,3-BPG to the C-2 position of 3-PG.

Reaction 8: Phosphoglycerate Mutase

Phosphoglycerate mutase (PGM) catalyzes a phosphoryl transfer, moving the phosphoryl group of 3-PG from C-3 to C-2 to form 2-Phosphoglycerate (2-PG). Mutases are enzymes that catalyze the migration of a functional group within a substrate molecule. PGM requires catalytic amounts of 2,3-BPG to maintain an active-site histidine residue in a phosphorylated form.

Reaction 9: Enolase

Enolase catalyzes a dehydration reaction (removal of a water molecule) to form phosphoenolpyruvate (PEP), creating a high-energy phosphate. Enolase requires either $Mg^{2+}$ or $K^+$ for activity.

Reaction 10: Pyruvate Kinase

Pyruvate kinase (PK) transfers a phosphoryl group from PEP to ADP, forming pyruvate and ATP. This second ATP-synthesizing reaction requires $Mg^{2+}$ and is stimulated by $K^+$. $\Delta G$ in erythrocytes is -23.0 kJ/mol. The enol tautomer of pyruvate spontaneously converts to the more stable keto form.

PK is allosterically activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine. Liver PK is regulated by covalent modification through hormones such as glucagon, which activate cAMP-dependent protein kinase. This kinase transfers a phosphoryl group from ATP to the enzyme. The phosphorylated form of PK is more strongly inhibited by ATP and alanine.

18.5 Metabolic Fates of NADH and Pyruvate

NADH can be recycled by both aerobic and anaerobic paths, each of which results in further metabolism of pyruvate. Under aerobic conditions, NADH is oxidized to $NAD^+$ in the mitochondrial electron-transport chain. Under anaerobic conditions, NADH is oxidized by lactate dehydrogenase or alcohol dehydrogenase, providing additional $NAD^+$ for glycolysis.

Anaerobic metabolism of pyruvate leads to lactate or ethanol:

• In yeast, pyruvate is reduced to ethanol (alcoholic fermentation).
• In other microorganisms and in animals, pyruvate is reduced to lactate (lactic acid fermentation).

Fermentation produces ATP, and organic molecules function as donors and acceptors of electrons.

Alcoholic Fermentation

Pyruvate is decarboxylated to acetaldehyde by pyruvate decarboxylase in an essentially irreversible reaction. Then, the reduction of acetaldehyde to ethanol by NADH is catalyzed by alcohol dehydrogenase.

Lactate Accumulation

Pyruvate is reduced to lactate by lactate dehydrogenase (LDH). Large amounts of ATP are generated rapidly, at the expense of lactate accumulation.

18.6 Regulation of Glycolysis

Under cellular conditions, the $\Delta G$ values for most steps in glycolysis are near zero except at Reactions 1, 3, and 10 (hexokinase, PFK, and PK reactions), which have large negative $\Delta G$ values. These three irreversible reactions are the primary sites of regulation for turning glycolysis on/off.

18.7 Alternative Substrates in Glycolysis

Other sugars, both simple and complex, can enter the cycle if they can be converted by appropriate enzymes to one of the intermediates of glycolysis.

Fructose Catabolism

Dietary fructose is degraded almost exclusively in the liver in an unregulated process that bypasses the PFK reaction. In the liver, fructose is phosphorylated at C-1 by the enzyme fructokinase.

Mannose

Mannose is phosphorylated by hexokinase and the mannose-6-phosphate produced is converted to F-6-P by phosphomannoisomerase.

Galactose

Galactose is phosphorylated from ATP at the C-1 position by galactokinase via the Leloir pathway. Galactosemia involves defects in galactose-1-P-uridylyltransferase (GALT).

Lactose Intolerance

Lactose intolerance is an inability to digest lactose due to the absence of the enzyme lactase in the intestines of adults.

Glycerol

Decomposition of triacylglycerols produces glycerol. Glycerol can also enter glycolysis:

1. Glycerol kinase phosphorylates glycerol using ATP to form sn-Glycerol-3-phosphate.
2. Glycerol-P dehydrogenase oxidizes sn-Glycerol-3-phosphate with $NAD^+$ to form Dihydroxyacetone phosphate (DHAP).