LE2

• Lecture 5 covers Carbohydrate Metabolism and Glycolysis

Historical Perspectives

• Winemaking and bread making were early applications of carbohydrate metabolism
• Louis Pasteur discovered that fermentation was caused by microorganisms
• Eduard Buchner showed that cell-free yeast extracts could perform fermentation
• Scientists found reagents that inhibit pathway products, leading to the identification of pathway intermediates
• The complete glycolysis pathway was elucidated in 1940 as the Embden-Meyerhof-Parnas Pathway

Glycolysis Overview

• Glycolysis, from the Greek words for sweet and splitting, means the breakdown of glucose
• It is nearly universal in living cells
• Glycolysis is the most completely understood pathway, including its regulation
• It performs a central metabolic role, generating both energy and metabolic intermediates for other pathways

Aerobic Glycolysis

• Pyruvate is oxidized to acetyl-CoA
• NADH is reoxidized in the mitochondria, enabling additional energy production
• Oxygen serves as the terminal electron acceptor

Fermentation

• Energy is yielded
• There is no net change in the oxidation state of the products compared to the substrates
• Homolactic fermentation is an example of fermentation

Chemical Strategy of Glycolysis - Strategy 1

• The first strategy involves adding phosphoryl groups to glucose, creating compounds with low phosphoryl group-transfer potential
• These phosphorylated compounds generally cannot be directly utilized as a free energy source by cells
• These compounds are important because they can be converted into phosphorylated compounds with high energy potential using ATP

Chemical Strategy of Glycolysis - Strategy 2

• The second strategy chemically converts low phosphoryl group-transfer potential compounds into those with high phosphoryl group-transfer potential
• As glycolysis proceeds, these compounds are further phosphorylated to generate high phosphoryl group-transfer potential, also using ATP
• The first half of glycolysis is an ATP investment phase, utilizing ATP to produce compounds with both low and high group-transfer potential

Chemical Strategy of Glycolysis - Strategy 3

• The third strategy, chemically coupling the energy-yielding hydrolysis of high-phosphoryl group-transfer potential compounds to the synthesis of ATP
• This is also known as substrate-level phosphorylation

Energy Investment Phase

• The energy investment phase constitutes the first half of glycolysis
• Two molecules of ATP are utilized to produce compounds with low and high group-transfer potential

Energy Generation Phase

• The second half of glycolysis is the energy generation phase
• Two important intermediate steps generate ATP via substrate-level phosphorylation, using NAD as an electron carrier
• Each glucose splits into 2 triose phosphates, generating 2 ATP per triose phosphate
• The net ATP generated in one pass of glycolysis is 2 ATP

Important Oxidation Step

• There is an important oxidation step in the second half of glycolysis that uses NAD as an electron carrier or reservoir
• NAD+ is reduced to NADH, which is then shuttled to the mitochondria for oxidative phosphorylation
• Approximately 2.5 ATP are produced for every mole of NADH

Reaction 1: The First ATP Investment

• Glucose is converted to Glucose-6-Phosphate (G-6-P) via Hexokinase
• ΔG = -18 kJ/mol
• This step involves the first ATP investment and also the first committed step in glycolysis
• Hexokinase catalyzes this reaction
• There are different isoforms of this enzyme depending on the tissue (e.g., liver, skeletal muscle)
• Phosphorylation requires ATP as the phosphate donor and free energy source
• The hydrolysis of the phosphoester bond between the gamma and beta phosphates of ATP makes this thermodynamically unfavorable step favorable with ΔG = -18.4 kJ/mol
• Mg2+ is essential for chelating the phosphoryl groups of ATP, positioning them for nucleophilic attack by hydroxyl groups

Hexokinase as 'Glucostat'

• Hexokinase acts as a "glucostat," sensing blood glucose levels, typically between 5-10 millimolar
• Orchestrates events based on physiological glucose levels
• It has broad specificity, phosphorylating glucose and other 6-carbon sugars like galactose
• It has low Km (0.01 - 0.1 mM), indicating high substrate affinity
• It sequesters G6O into the cell, requiring high substrate concentration

Hexokinase Detail

• Hexokinase is made of 2 domains which catalyzes the phosphorylation of glucose.
• Its activity depends partly on the presence of Mg2+
• At least 5 isoforms of hexokinase exist depending on the tissue type
• Skeletal muscle, being highly metabolically active, utilizes Hexokinase 1
• Hexokinase 1 has a low Km (0.04mM), far below normal physiological levels of glucose (5-10 mM)
• HK1 effectively sequesters glucose for its use, given skeletal muscle's high metabolic activity driving mechanical work.
• HK1 remains active even when glucose levels drop to normal physiological levels, constantly supplying ATP for contraction and movement
• HK1 phosphorylates glucose into glucose-6-phosphate (G6P), trapping it inside for glycolysis and ATP production once it enters muscle cells

Hexokinase 4

• Liver cells, less metabolically active, utilize Hexokinase 4
• The liver is a major organ for glycogen storage
• Hexokinase 4 has high Km (7.5 mM); even at normal glucose physiological levels, its maximal velocity is only half of hexokinase 1, so it can spare glucose for other tissues that have high metabolic demands
• Hexokinase 4 will sequester glucose only when the body is overwhelmed with sugar
• HK4 is mostly inactive when blood glucose levels are low (e.g., fasting or intense exercise), because it doesn't effectively bind glucose at concentrations below its Km.
• HK4 becomes active when glucose levels rise significantly (e.g., after eating), allowing the liver to store excess glucose as glycogen

Comparing Hexokinases

• The difference between 2 types of hexokinase is their affinity for glucose
• Hexokinase 1: low(0.04mM) -> high affinity
• Hexokinase 4: High(7.5mM) -> low affinity
• Hexokinase 1: Active -> ensures glucose uptake
• Hexokinase 4: Inactive -> spares glucose for other tissues
• Hexokinase 1: Always active
• Hexokinase 4: Becomes active only when glucose is high.
• Hexokinase 1 Function: Muscle energy production
• Hexokinase 4 Function: Regulates glucose storage in the liver

Reaction 2: Isomerization of G6P

• Catalyzed by Glucose-6-phosphate isomerase
• Glucose-6-Phosphate is converted to its keto form Fructose-6-Phosphate.
• Involves isomerization of an aldehyde to its keto counterpart
• It is a fairly endergonic reaction (ΔG = +1.7kJ/mol)

Reaction 3: The Second ATP Investment

• Fructose-6-P becomes Fructose-1,6-Bisphosphate via Phosphofructokinase
• ΔG= -15kJ/mol
• Fructose-1,6-Bisphosphate does not have high energy to synthesize energy
• Thus synthesis aims preparing this compound for splitting or lysis, for glycolysis to occur

Role of PFK

• PFK is a major control point in glycolysis
• For prevent futile energy cycle, in glycolysis with a control point
• A number of glyolysis intermediates, exert their regulatory role for this enzyme

Regulation of PFK

• In Step 3, ATP inhibits PFK-1
• High ATP concentration signifies high energy status, inhibiting glycolysis for stop or inhibit
• Therefore, ATP amount inhibits PFK so slow then, glucose is not over produced from ATP
• ADP level high signals low energy which activates this enzyme
• If AMP is high, also signals low energy level activating PFK-1 for activate glycolysis
• Citrate is the intermidiate for critic acid synthesis, and inhibits PFK, during high energy status
• Activate glyocolysis and will switch off gluconeogenesis
• When AMP is high energy is low, then glycolysis activates

Significance of PFK

• The same substrate/allosteric inhibitor will inhibit the enzyme fructose bisphosphatase, acting as opposite to PFK-1
• Fructose bisphosphatase dephosphorylates or removes the phosphate from Fructose-1,6- Bisphosphate and converts it to Fructose-6-Phosphate
• PFK plays a central role in controlling glycolysis, due to the catalysis of one of the pathway's rate-determining step
• enhanced allosterically by including AMP
• inhibits allosterically by other substanced including ATP and citrate

Reaction 4: Cleavage of F-1,6-BP to Triose Phosphates

• The enzyme Aldolase catalyzes a reaction to divide Fructose-1,6-bisphosphate
• Results in Dihydroxyacetone Phosphate (DHAP) and Glyceraldehyde-3-Phosphate (GAP) - ΔG= +23.9kJ/mol
• considered high energy level compoound but, to recover ATP

Action of Enzyme Aldolase

• A reaction cleaves Fructose-1,6-bisphosphate into (1) Dihydroxyacetone Phosphate/DHAP and (2) Glyceraldehyde-3-Phosphate/GAP
• normal physiological process, very high intermediates to make the only time reaction process is reversible to generate
• under physiological standards never truly occurs

Reaction 5: Isomerization of DHAP

• GAP then Dihydroxyacetone Phosphate catalyzed with Triosephosphate Isomerase/TPI ΔG= 7.6kJ/mol
• Interconversion or isomerization occurs for GAP and DHAP, a highly unstable
• catalyzed via Triose Phosphate Isomerase / Triosephosphate Isomerase however thermodynamically unfavorbale which, production of GAP is higher than DHAP for favorable under phys conditions, slight
• GAP has more affinity instead of DHAP reaction for the next enxyme.
• DHAP converts to GAP
• 1 ml, fructose- 1 has a double of every since product is made