Glycolysis And Gluconeogenesis

Lecture 14. Glycolysis and gluconeogenesis • Glycolysis • Break down the glucose • Gluconeogenesis • Form the glucose (usually new formation of glucose) • Regulation of Glycolysis and Gluconeogenesis

Glycolysis • (Greek: glykus = sweet, lysis = loosening) • Glycolysis is a sequence of ten enzymatic reaction in which one molecule of glucose is converted to two molecules of pyruvate and ATP • Glycolysis occurs in two stages – Each stage consists of 5 reactions

First Stage in Glycolysis Reaction 1 The first step in glycolysis is the transfer of the gamma phosphate group of ATP to the hydroxyl group on C6 of glucose to form glucose-6-phosphate (G6P). This reaction is catalyzed by hexokinase.

Phosphorylation of glucose traps it intracellularly because the negative charges of the phosphate group prevents any possibility of diffusing through the membrane.

One ATP consumed

First Stage in Glycolysis Reaction 2 G6P is converted to fructose-6-phosphate (F6P) by phosphoglucose isomerase.

This is the isomerization of an aldose to a ketose.

First Stage in Glycolysis

First Stage in Glycolysis Reaction 3 Phosphofructokinase (PFK) catalyzes the phosphorlyation of F6P to yield fructose-1,6-biphosphate (FBP).

Hydroxyl group is phosphorylated

 a kinase Another ATP molecule consumed

First Stage in Glycolysis Reaction 4

Cleavage of 6 carbon glucose into 2 fragments: DHAP and GAP

Aldolase catalyzes the cleavage of FBP to form the trioses glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP).

The numbering system changes. Atoms 1, 2, and 3 of fructose become atoms 3, 2, and 1 of DHAP. Atoms 4, 5, and 6 become atoms 1, 2 and 3 of GAP. Reaction 4 is an aldol cleavage (or a retro aldol condensation) with the following mechanism.

First Stage in Glycolysis An aldol is derived from an aldehyde and an alcohol product.

Note: Aldol cleavage between C3 and C4 of FBP requires a carbonyl at C2 and a hydroxyl at C4. Thus, the purpose of Reaction 2 where G6P is isomerized to F6P becomes clear. Aldol cleavage of F6P yields two interconvertible C3 compounds. Aldol cleavage of G6P would yield two and four carbon compounds as shown below.

First Stage in Glycolysis Reaction 5

Isomerization by triose phosphate isomerase

Triose phosphate isomerase catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to glyceraldehydes-3phosphate, which continues along the glycolytic pathway.

Review of the first stage of Glycolysis • No ATP molecules so far have been made • Two ATP have been consumed

The Second Stage of Glycolysis Reaction 6 Glyceraldehyde-3-phosphate dehydrogenase catalyzes the oxidation of the aldehyde of glyceraldehyde-3-phosphate by NAD+ and the subsequent phosphorylation to form the acyl phosphate 1,3 bisphosphoglycerate (1,3-BPG).

The Second Stage of Glycolysis NAD+ is derived from the vitamin niacin. A deficiency of niacin causes the disease pellagra.

The Second Stage of Glycolysis Reaction 7 The phosphoryl group on C1 of 1,3-BPG is transferred to ADP to make ATP and 3-phosphoglycerate (3PG). 1,3-BPG is very unstable because it is an acyl phosphate.

1,3-BPG + H2O

3PG + Pi

ΔG = -11.8 kcal/mole

This reaction is catalyzed by phosphoglycerate kinase. Because two molecules of GAP entered the 2nd stage of glycolysis, two molecules of ATP are synthesized. Since two molecules of ATP were consumed in the first stage, at this point in glycolysis, the net synthesis of ATP is zero. This means actually 2 ATP molecules are generated from one glucose

Tissue Specific Metabolism In Erythrocytes a Detour from Glycolysis (Rapopart-Luebering Shunt)

Rough idea

2,3-BPG binds preferentially to the T-state of Hb thereby decreasing oxygen affinity. Concentration in the RBC is equivalent to Hb. If it was not present in RBC Hb would not release O2 and instead Hb would be an O2 trap. Concentration is increased under hypoxic conditions such as emphesyma and high altitudes.

The Second Stage of Glycolysis Reaction 8 3-phospoglycerate is converted to 2-phosphoglycerate by phosphoglycerate mutase.

The Second Stage of Glycolysis Reaction 9 Enolase catalyzes the dehydration of 2-phosphoglycerate to phosphoenolpyruvate.

Like 1,3-BPG, phosphenolpyruvate is unstable.

PEP + H2O

pyruvate + Pi

ΔG = -14.8 kcal/mole

The Second Stage of Glycolysis Reaction 10 The phosphoryl group of PEP is transferred to ADP to make ATP and pyruvate.

Net synthesis of ATP is two.

Review of the Second Stage of Glycolysis

The Fate of the End Product of Glycolysis: Pyruvate

Fermentation (anaerobic biological process) In yeast under anaerobic conditions, the NAD+ needed for glycolysis is regenerated by converting pyruvate into ethanol and CO2 by two enzymes.

The ethanol of course can be purified by distillation and used for a variety of activities. The CO2 is used to leaven bread.

Fermentation (anaerobic biological process) Lactic acid formation Under anaerobic conditions in muscle, pyruvate is converted to lactate.

Clinically, measurement of blood lactate levels can be used to monitor the metabolism of a patient. Lactate is a sensitive indicator of inadequate tissue oxygenation. If blood lactate is high, this indicator either a decrease in oxygen delievery or oxygen consumption by tissues.

Gluconeogenesis

Synthesis of glucose

The ability to synthesize glucose is essential for the survival of humans and other animals. This is because the brain, red blood cells, kidney, liver, cornea and testis use glucose as their sole or major fuel source. In fact, the human brain requires over 120 grams of glucose / day.

Gluconeogenesis Gluconeogensis maintains the blood glucose level long after all dietary glucose has been absorbed and oxidized, and glycogen stores depleted.

Gluconeogenesis occurs primarily in the liver. The produced glucose is excreted into the blood to supply other tissues.

Gluconeogenesis Most of the reactions of gluconeogenesis are glycolytic reactions going in reverse (bc those enzymes can catalyze both the forward and reverse reactions). However, the reactions catalyzed by hexokinase, phophofructokinase and pyruvate kinase have large negative free energy changes (i.e. the equilibrium lies far towards the products for glycolysis). As a result, the reactions are replaced by reactions that make glucose synthesis thermodynamically favorable.

Gluconeogenesis

Ethanol Consumption Causes Hypoglycemia!

NADH builds up in the body; There’s not enough starting material for gluconeogenesis

Regulation of Glycolysis and Gluconeogenesis Need to regulate glycolysis and gluconeogenesis to prevent futile cycling.

Different Levels of Metabolic Regulation Availability of substrates can regulate the metabolism in seconds to minutes (very fast) without substrates the reaaction will stop Allosteric activators or inhibitors will regulate the metabolism in minutes (very fast) because these compounds inhibit very quickly Covalent modification of enzymes – much slower because you have to go through covalent bond formation – minutes to hours, later we will see this extending through phosphorylation or dephos. Synthesis of enzymes takes hours, even days to regulate.. Remember there are 4 different levels

Allosteric Control Phophofructokinase (PFK-1) is the major point controlling flux through the glycolytic pathway. PFK-1 is subject to allosteric activation and inhibition. This type of regulation is short term and only regulates the metabolism of the individual cell. Green triangle: activator Red x: inhibitor

ATP acts as an allosteric inhibitor of PFK, while ADP and AMP act as allosteric activators.

Allosteric Control PFK-1 is a homeotetramer that exists in T and R states. Each subunit has two binding sites for ATP, the active site and an allosteric site. The allosteric site is only accessible in the T state.

Allosteric Control When ATP levels are low. ATP binds in the active site along with F6P. F6P bind prefentially to the R state, shifting the equilibrium to the R state. This results in a hyperbolic activity curve. When ATP levels are high, ATP binds in the allosteric site (changes equilibrium to inactive state) This pushes the equilibrium towards the inactive T state, resulting in a sigmodial activity curve.

If this is how glycolysis and gluconeogenesis are regulated at the cellular level then how are they regulated at the organismal level?

Hormonal Control Insulin and Glucagon Glucagon secreted by alpha cell Insulin secreted by beta cell

Hormonal Control Insulin and Glucagon

Insulin Connected by 2 disulfide bonds to form dimer

Insulin

Glucagon

Epinephrine is a hormone secreted by the adrenal medulla upon stimulation by the central nervous system in response to stress, as anger or fear, and acting to increase heart rate, blood pressure, cardiac output, and carbohydrate metabolism. Glucagon is a hormone secreted by the pancreas that acts in opposition to insulin in the regulation of blood glucose levels

dimer

Have an idea about cyclic AMP structure

Putting it all together

Glucagon also Phosphorylates Pyruvate Kinase

Clinical Correlation Glycolysis and Oncology The rate of glycolysis is about 10X faster in solid tumors that in normal tissue. This is called the Warburg effect. This is because solid tumors are hypoxic due to an insufficient capillary network. As a result, solid tumors depend on anaerobic glycolysis to generate ATP. The increased rate of glycolysis in cancer cells has led to a new and sensitive method in cancer diagnosis and assessment. The patient is administered 2-deoxy-2-[18F]-fluoro-D-glucose (FDG) intravenously. FDG is transported into cells by GluTs. It is readily phosphorylated by hexokinase. However, phosphorylated FDG is not a substrate for phosphoglucose isomerase because the OH group at C2 is missing. CH2OH O H H OH OH

H

OH H

18

F

Clinical Correlation Thus FDG accumulates in cancer cells. It is not known if the increased glucose accumulation is due to Increase in GluT activity Increase in hexokinase activity Decrease in phosphatase activity Some combination of the above Since 18F is radioactive with a half-life of 2 hours, FDG is detected positron emission tomography (PET) The technique is used for Detection and staging of primary tumors Detection of metastasis Differentiation of malignant form benign tumors

Homework Questions • Describe energy change in two different metabolisms • Name three purposes of the energy generated by catabolic pathways • If radio-labeled C-1 of glucose is used for glycolysis and alcoholic fermentation, which carbon in the final product(s) is labeled • Describe how ethanol consumption causes hypoglycemia • Describe how the amount of ATP affects activity of phosphofructokinase-1 (PFK-1)