Lecture 16 Glycolysis

Glycolysis Occurs…?

in the cytoplasm of all cell types

RBCs and Cells w/o mitochondria

…fully rely on glycolysis for energy production

Hemolytic Anemia

inability to carry out glycolysis

Major Carbs in Human Diet

starch, sucrose, lactose, fructose, and glucose

Starch

polymers of glucose liner alpha (1-4) bonds, branch alpha 1,6 bonds more common in glycogen

Carb Digestion Steps

1) starches broken down by salivary alpha amylase, hydrolysis of alpha 1,4 glycosidic bonds

2) pancreatic alpha amylase digestion, hydrolysis of alpha 1,4 glycosidic bonds

3) produce alpha maltose, alpha isomaltose, tri/oligosaccharides, and dextrins

Maltose

disaccharide of glucose linked by alpha 1,4 bond broken by maltase on surface of brush border of intestinal epithelial cells into glucose

Isomaltose

disaccharide of glucose linked by alpha 1,6 bond broken by isomaltase on surface of brush border of intestinal epithelial cells into glucose

Sucrose

disaccharide of glucose linked to fructose in alpha 1,2 linkage broken by sucrase on surface of brush border of intestinal epithelial cells

Lactose

disaccharide of galactose and glucose by beta 1,4 linkage, broken down by lactase into glucose and galactose on surface of brush border of intestinal epithelial cells

Lactase Absence

lactose intolerance treatment can be reducing consumption of milk, lactase products

4 Steps of Glucose Absorption

1) SGLT1 symporter with glucose and galactose against gradient, energy provided by electrochem gradient of Na going high to low, on apical border of intestinal cells

2) Na K antiporter uses ATP to move Na from low to high, keeps cytoplasm levels low, helps SGLT1 operation

3) GLUT2 glucose transporter of glucose from high to low, on basolateral side, high capacity Vmax but low affinity high Km for glucose, moves high glucose quickly

4) GLUT5 facilitated fructose transporter on apical border

SGLT1 Deficiency

causes glucose and galactose malabsorption

GLUT5 Deficiency

fructose malabsorption (dietary fructose intolerance)

2 Stages of Glycolysis

investment and payoff stages

Energy Investment phase of Glycolysis

uses 2 ATP molecules of ATP/glucose but makes no ATP, traps and prepares glucose for oxidation steps

Glucose converted to glyceraldehyde 3-phosphate GAP, 5 Steps

1) phosphorylation of glucose

2) isomerization

3) second phosphorylation of fructose-6P (committed step)

4) cleavage into 2 3C molecules

5) isomerization of DHAP to GAP

Phosphorylation, Phosphoryl Group

acts as handle for enzyme recognition and provides increased binding free energy

Glycolysis Step 1 Molecules

glucose with hexokinase, ATP, and Mg2+ makes glucose 6-phosphate, exergonic -16.7 kj/mol

Phosphorylation in Glycolysis, Cleft Closing

glucose binds first, cleft closing dehydrates active site preventing nucleophilic attack by water and ATPase, NP binding site excludes water, facilitates aspartate to act as back and start reaction

Hexokinase Reaction (Phosphoryl Group Transfer) 1st step

1) C6-OH deprotonated by aspartate (base) and becomes an aspartic acid and a nucleophile

2) charged C6-O- attacks gamma P

3) covalent bond intermediate between C6-O and P

4) phosphoanhydride bond between beta and gamma phosphates broken, good LG

Hexokinase I

irreversible but regulatory step, low Km and lower vmax (low capacity), permits efficient phosphorylation of glucose even at low concentration

Hexokinase I inhibition

by Glucose-6-P, prevents from tying up all intracellular Pi in form of G-6-P

Hexokinase IV

glucokinase, expressed in hepatocytes and pancreatic b cells, high Km and Vmax (higher capacity), not inhibited by G6P

Isomerization, Step 2

conversation, of aldose to ketose sugar, endergonic, goal to convert 6C starting to 2 3C carbon units, carbonyl at 61 is moved to carbonyl at C2 to promote aldol reaction

What must happen first before isomerization?

the glucose 6-P must be converted to fructose 6-P

Phosphoglucose Isomerase Mechanism, 9 Steps

1) base deprotonates H2O to OH-

2) Lys organizes OH-

3) OH- deprotonated C1-OH, H2O

4) C5-O deprotonate His

5) ring opens

6) glutamate deprotonates C2-H, enediolate

7) enediolate deprotonates glutamic acid, ketone

😎 HIS deprotonates C5-OH

9) ring closes and forms fructose 6-phosphate

Glycolysis Step 3, Second Phosphorylation

fructose 6-phophate to fructose 1,6-biphosphate using second ATP and phosphofructokinase

Glycolysis Step 3 Characteristics

commited step, irreversible, cell can now split 1,6-BP to 2 trioses , allosterically regulated by ATP/AMP, most important regulatory point in glycolysis

Regulation of Phosphofructokinase

PRFK-1 inhibited allosterically by ATP “energy rich signal,” ATP binds at 2nd site away from catalytic site, AMP reverses inhibition by ATP

Fructose 2,6BP Regulator

High F6-P causes High PRK2 causes high F2,6BP causes high PFK1 activity increased affinity for F6-P

2 Effects of F2,6BP (made by PFK2) on PFK1

1) more enzyme PFK1 is active at lower concentrations in the presence of F2,6BP

2) ATP as a substrate stimulates reaction but as the concentration increases it acts as an allosteric inhibitor, this effect is reduced by F2,6BP and makes it less sensitive to ATP inhibition

Aldolase, Step 4

F1,6 BP uses aldolase to converte to dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phophate (GAP), 23.8 jk/mol

Aldolase Reaction Favorability

23.8 kJ/mol is energetically uphill but the products are rapidly deleted so the reaction is pulled forwards

Step 5, Isomerization of DHAP and G3-P

DHAP converted to glyceraldehyde 3-phosphate using triose phosphate isomerase, G= 7.5 kJ/mol

Step 5 Isomerization Characteristics

ketose-aldose isomerase, isomerization produces 2 molecules of G3-P, continual metabolism of G3-P in glycolysis drives the reaction forward

What is the end of the investment phase?

step 5, isomerization of DHAP and G3P

Energy Generation Phase of Glycolysis

2 3C units oxidized to produce pyruvate, 4 ATP, and 2NADH

G3P is converted to pyruvate by which 5 steps?

1) oxidation of G3P to 1,3BPG

2) phosphorylation of ADP

3) mutase, conversion of 3PG to 2PG

4) dehydration by enolase

5) phosphorylation of ADP, gives pyruvate

Glycolysis Step 6, oxidation of G3P

G3P + NAD + Pi + G3P dehydrogenase gives 1,3-biphosphoglycerate (1,3-BPG), G= 6.3kJ/mol

Oxidation of G3P Characteristics

doesn’t use ATP, oxidation of G3P powers formation of 1,3 BPG which has high phosphoryl transfer potential, multi-step phosphorylation

1,3 BPG

an acyl phosphate, a mixed anhydride of phosphoric acid and carboxylic acid (carbonyl)

Multi Step phosphorylation of G3P

starts off as aldehyde and oxidized to carboxylic acid by transferring hydride from NAD+ donor then coupled with dehydration to form acyl phosphate (energetically unfavorable)

G3P Dehydrogenase 7 Step Mechanism

1) base (His) deprotonates Cys thiol group

2) sulfur attacks aldehyde to make thiohemiacetal

3) unstable oxyanion collapses

4) hydride transferred to NAD+ and leaves a thioester bond

5) Pi attack carbonyl and makes tetrahedral oxyanion

6) oxyanion collapses to sever thioester

7) base deprotonated

Thioester is…?

higher energy than carboxylic acid, can’t resonate and less stable, easier to cleave

Without thioester

reaction of stable carboxylate with phosphate

With thioester

energy trapped in thioester

Step 7 Phosphoglycerate kinase

1,3 BPG with phosphoglycerate kinase and Mg makes 3-phosphoglycerate and generated ATP

3-phosphoglyerate Characteristics

Lys 29 guides C1P to gamma position of ATP, substrate level phosphorylation , energy released in oxidation of aldehyde to carboxylate conserved through ATP formation

Glycolysis step 8, shift of phosphate group from C3 to C2

3-phosphoglycerae with phosphoglycerate mutase and Mg make 2-phosphoglycerate, G= 4.4 kJ/ mol, reaction uses 2 separate phosphate

Mutase

applied to enzymes that catalyze migration of functional groups from our position to another in same substance molecule

Step 8 Phosphoglycerate Mutase

allosteric shape change, alleviate crush of pancreas

Step 8 phosphorylation Mutase Stemp

1) His deprotonared C2Oh aeggevt to attack protein complex Phospo-his

2) CO2 oxyanion gets Pi from Phospho-His, makes 2,3-biphosphoglycerate

3) dephosphorylated His takes Pi from C3-Pi makes 2 phosphoglycerate

Glycolysis Step 9 Dehydration of 2-phosphoglycerate

1-phosphoglycerate with enolase and water leaves makes phosphoenolpyruvate, 7.5 kJ/mol

Dehydration of 2,phosphoglycerate Characteristics

enolase catalases reversible dehydration of 2-phosphoglycerate to create phospho-enol pyruvate, keeps high phosphoryl group transfer potential

Step 10 Pyruvate Kinase

phosphoenolpyruvate with Md, K, and ADP to form pyruvate (2 forms, enol and keto)

Pyruvate Kinase Unique characteristics

ATP regeneration rep, substrate level phosphorylates, irreversible and regulated reactions

3 Regulates od Pyruvate in Lab

allosteric activator F 1,6 BP, allosteric inhibitor AMP, protein phosphorylation

Pyruvate Kinase Deficiency

not enough ATP for RBC survival, removed by the spleen, most common cause of hereditary anemia, build up of 2,4-biphosphoglycerate which helps release O2 to tissue

Aerobic Vs Anaerobic Glycolysis

1) Nad+ and NADH metabolizes in feweer minutes

2) NADH may be rapidly oxidized for NAD+ glycolysis to happen

3) in aerobic glycolysis, reduce e power of NADH transferred to mitochondria by malate-aspartate and G3P shuttles regenerating Nad_

4) in in anaerobic glycolysis, NADH oxidized to Nad+ by lactate dehydrogenase

7) uin aerobic, 30-32 ATPs/glucose produced, in anaerobic, only 2 ATP produced