Glycolysis: Preparatory Phase

Glycolysis Overview

• Also known as the Embden-Meyerhof-Parnas pathway.
• Glucose enters most cells via specific carriers from the extracellular matrix to the cytosol.
• All glycolytic enzymes are in the cytosol.
• Glucose is converted to two 3-carbon molecules (pyruvate).
• Glycolysis has 10 reactions divided into the preparatory and payoff phases.

Preparatory Phase of Glycolysis

• Requires energy investment.
• Two ATP molecules are invested per glucose molecule.
• Glucose is phosphorylated and cleaved to form two glyceraldehyde-3-phosphate molecules.
• Consists of the phosphorylation of glucose, isomerization of glucose-6-phosphate to fructose-6-phosphate, phosphorylation of fructose-6-phosphate, cleavage of fructose-1,6-bisphosphate, and interconversion of triose phosphates.
• Step 1: Glucose is phosphorylated at C-6 to yield glucose-6-phosphate, requiring ATP and catalyzed by hexokinase.
• Step 2: GGlucos-6-phosphate is isomerized to fructose-6-phosphate, catalyzed by phosphohexose isomerase.
• Step 3: Fructose-6-phosphate is phosphorylated at C-1 to fructose-1,6-bisphosphate, catalyzed by phosphofructokinase-1, requiring ATP, and is the first committed step in glycolysis.
• Step 4: Fructose-1,6-bisphosphate is cleaved in a reaction catalyzed by aldolase, from which the process derived the name glycolysis.
• Step 5: Dihydroxyacetone phosphate is isomerized to glyceraldehyde-3-phosphate, catalyzed by triose phosphate isomerase.

Payoff Phase of Glycolysis

• Energy is recovered
• Two pyruvate molecules produced per glucose.
• Four ATP molecules are produced per glucose molecule.
• Step 6: The aldehyde group of glyceraldehyde-3-phosphate is oxidized, donating an H+ to NAD+ to form NADH+H+, catalyzed by glyceraldehyde-3-phosphate dehydrogenase.
• Step 7: The phosphoryl group attached to 1,3-bisphosphoglycerate is transferred to ADP, generating ATP, catalyzed by phosphoglycerate kinase.
• Step 8: The phosphoryl group attached to C-3 of 3-bisphosphoglycerate is shifted to C-2 of 2-phosphoglycerate, requires magnesium, and is catalyzed by phosphoglycerate mutase.
• Step 9: Water is removed from 2-phosphoglycerate, catalyzed by enolase, which requires magnesium.
• Step 10: The phosphoryl group of phosphoenolpyruvate is transferred to ADP, generating ATP and pyruvate, catalyzed by pyruvate kinase.

General Equation of Glycolysis

• Glucose + 2NAD+ 2ADP + 2Pi → 2 pyruvate + 2NADH + 2H+ + 2ATP + 2H₂O

Regulation of Glycolysis

• Allosteric regulation occurs at key points in the pathway to maintain constant ATP levels and intermediate concentrations at: Step 1, Step 3 and Step 10.
• Glycolysis is hormonally regulated by insulin, glucagon and epinephrine.

ATP Net Gain from Glycolysis

• Glucose + 2ATP + 2NAD+ 4ADP + 2Pi → 2 pyruvate + 2ADP + 2NADH + 2H+ 4ATP + 2H₂O

Feeder Pathways for Glycolysis

• Feeder pathways are minor routes that supply intermediates to major metabolic pathways.
• Glycogen, starch, disaccharides, and other hexoses can act as sources of intermediates for glycolysis.

Catabolism of Glycogen and Starch

Glycogen

• Glycogen enters Glycolysis in two steps:

1. Glycogen breaks down to glucose-1-phosphate using phosphorylase
2. Glucose-1-phosphate is converted to glucose-6-phosphate using phosphoglucomutase

Starch

• Starch enters Glycolysis in one step:

1. Starch breaks down to glucose using α-amylase

Catabolism of Dietary Disaccharides

Lactose

• Lactose enters glycolysis via two paths:
  1. Lactose is broken down to glucose and galactose using lactase
     • Glucose directly enters glycolysis
     • Galactose is phosphorylated then isomerized to UDP-glucose

Sucrose

• Enters glycolysis using two paths:
  1. Sucrose is broken down into glucose and fructose using sucrase
     • Glucose directly enters the first step of glycolysis
     • Fructose is phosphorylated to Fructose-1-phosphate using fructokinase

Catabolism of Other Hexoses

Mannose

• This enters Glycolysis in two steps:
  1. Mannose is phosphorylated to mannose-6-phosphate using ATP and hexokinase
  2. Mannose-6-phosphate is converted to fructose-6-phosphate using phosphomannose isomerase

Metabolic Fates of Pyruvate

• It depends on the availability of oxygen.
• Early Earth lacked oxygen, thus primitive organisms produced energy from fuel molecules (ATP, NADH, pyruvate) in anaerobic conditions.
• Most organisms produce energy in aerobic and anaerobic conditions.

Aerobic Fate of Pyruvate: CO₂ and H₂O

• Pyruvate is completely oxidized to CO2 and H2O via the citric acid cycle under aerobic conditions.
• Pyruvate first loses its carboxylate group as CO2, forming the acetyl group of acetyl-CoA.
• The acetyl group is then oxidized through the citric acid cycle to CO2.
• Electrons from these reactions are passed to O2 to form H2O.

Anaerobic Fates of Pyruvate: Lactic Acid Fermentation

• During hypoxia and/or vigorous physical activity, oxygen is rapidly consumed and pyruvate can no longer be oxidized in the carboxylic acid cycle.
• Animal skeletal muscles reduce excess pyruvate to lactate using lactate dehydrogenase to regenerate NAD+.
• Excess lactate is transported back to the liver to be resynthesized to glucose.

Lactic Acid Fermentation

• C=O
• CH3
• Pyruvate
• NADH+H /NAD
• HO-C-H
• CH3
• L-Lactate

Anaerobic Fates of Pyruvate: Ethanol Fermentation

• Yeasts and microorganisms ferment glucose to ethanol instead of lactate.
• Acetaldehyde is formed by removing the carboxyl group of pyruvate using pyruvate decarboxylase (requires Mg2+ and thiamine pyrophosphate).
• Acetaldehyde is reduced to ethanol using alcohol dehydrogenase.

Gluconeogenesis

• Occurs:
  • When dietary glucose sources are unavailable.
  • When the liver and kidney exhaust their glycogen stores.
• Goal: Convert pyruvate to glucose.
• The regulated steps of glycolysis must be bypassed, which requires large amounts of energy.
• STEP 1: Needs two sequential reactions

Step 1 Reaction 1

• Pyruvate is first transported from the cytosol to the mitochondria for reaction.
• Pyruvate is converted to oxaloacetate using pyruvate carboxylase (requires ATP).
• Biotin acts as a bicarbonate carrier.

Step 1 Reaction 2

• Oxaloacetate cannot be transported directly back to the cytosol; therefore, it is first converted to malate, then reoxidized to oxaloacetate (using NAD+) once outside the mitochondria.
• Once in the cytosol, oxaloacetate is converted to phosphoenolpyruvate (PEP) using phosphoenolpyruvate (PEP) carboxykinase (requires GTP).

Bypass Reactions of Gluconeogenesis

Step 2: The Conversion of Fructose-1,6-Bisphosphate to Fructose-6-phosphate

• Fructose-1,6-Bisphosphate is hydrolyzed to Fructose-6-phosphate using the enzyme fructose-1,6-bisphosphatase
• The enzyme needs Magnesium to function

Step 3: The Conversion of Glucose-6-phosphate to Glucose

• Glucose-6-phosphate is hydrolyzed to Glucose using the enzyme glucose-6-phosphatase
• The enzyme needs Magnesium to function

Pentose Phosphate Pathway

• Other Fate of Glucose: Ribose-5-Phosphate.
• While glycolysis is the major catabolic pathway undergone by glucose, some molecules needed by the cell cannot be provided by glycolysis alone.
• Many rapidly dividing cells need a constant supply of pentoses as precursors to important coenzymes.
• Some tissues require electron donors in the form of NADPH to synthesize antioxidants, and even hormones.
• NADPH comes from the anabolic oxidation of Glucose to Ribose-5-phosphate.

The Pentose Phosphate Pathway (Hexose Monophosphate Shunt)

• Can be subdivided in the nonoxidative and oxidative phase
• The Oxidative Phase produces the pentose phosphate, Ribose-5-phosphate and NADPH as products
• The Nonoxidative Phase recycles the unused Ribose-5-phosphate back to Glucose-6-phosphate.

Glycogenolysis

• Definition: The catabolic breakdown of glycogen.
• It Occurs:
  • When dietary glucose is unavailable (fasting, vigorous physical activity)
• Glycogen is broken down to provide glucose and can occur in skeletal muscle and the liver.
  • Muscle glycogen: Can be depleted in less than an hour.
  • Liver glycogen: Can provide energy for up to 12-24 hours.
• The breakdown of glycogen through glycogenolysis is different from the breakdown of glycogen from digestion.

Glycogenolysis - Steps

Step 1

• Simultaneous Removal and Phosphorylation of a Glucose from Glycogen.
• Glycogen phosphorylase is used to remove one glucose subunit at a time from the non-reducing end until it reaches the fourth glucose unit from the branch.
• The free glucose is simultaneously phosphorylated to form Glucose-1-phosphate.

Step 2

• Removal of Branches Using a Debranching Enzyme.
• Since glycogen phosphorylase can only cleave terminal glucose units, the branches of the glycogen molecule need to be broken.
• A glycogen debranching enzyme removes the branches of glycogen to make more glucose residues accessible to phosphorolysis.

Step 3

• Isomerization of Glucose-1-Phosphate to Glucose-6-Phosphate.
• The reversible isomerization of Glucose-1-phosphate to Glucose-6-phosphate, using the enzyme Phosphoglucomutase, occurs in two reactions: - Reaction 1: Glucose-1-phosphate is phosphorylated at C-6 - Reaction 2: The phosphoryl at C-1 is transferred to the enzyme, leaving behind Glucose-6-phosphate

Glycogenesis

• Is the conversion of glucose to glycogen.
• Happens when glucose isn't used.
• Requires free energy input, in the form of UTP, to proceed.

Glycogenesis Steps

• Step 1: Isomerization of Glucose-6-phosphate to Glucose-1-phosphate (phosphoglucomutase action is reversible) The formation of Glucose-6-phosphate is simply the reverse of the reaction for the Glucose-1-phosphate.
• Step 2: Conversion of Glucose-1-phosphate to the Sugar Nucleotide, UDP-Glucose Glucose-1-phosphate is converted to the sugar nucleotide, UDP-Glucose using UDP-Glucose phosphorylase (requires UTP)
• Step 3: Elongation of the Glycogen Chain Using the enzyme glycogen, the produced UDP-Glucose is attached to the nonreducing end of a glycogen chain. The enzyme also simultaneously removes the attached UDP non.
• Step 4: Formation of Branches Glycogen is a highly branched polysaccharide, synthesized from branches of a new glycogen strand. Once the glycogen chain reaches 11 residues in length, the glycogen branching enzyme removes 4-6 residues from the chain for step 1. The same enzyme transfers removed residues to C-6 of a glucose to form a branch in step 2.

Carbohydrate Digestion

Mouth

• Food is masticated in the oral cavity and turned into bolus.
• Saliva contains α-amylase, which hydrolyzes starch into maltose, and other smaller saccharides.
• Minimal absorption as food is swallowed quickly.

Stomach

• Salivary amylase denatures as a result of stomach acid.
• No carbohydrate digesting enzymes are present in the stomach.
• Swallowed food is turned into chyme.

Small Intestine

• Bile is released which neutralizes the pH of gastric juices.
• Pancreatic ά-amylase breaks down any remaining polysaccharide chains.
• All monosaccharides are absorbed.

Liver

• Monosaccharides travel to the liver and enter the bloodstream through the hepatic portal vein.
• Excess glucose is stored as glycogen.

Carbohydrates

• Are polyhydroxy aldehydes and ketones.
• Generally have the empirical formula (CH2O)n
• The most abundant class of biomolecules.
• Play a variety of roles in the biosphere Cellular Protection
• Cellulose of plant cell walls Energy Storage
• Potato starch granules Structural Components
• Chitin of shrimp shells