BIOS 301 - Glycolysis

Homo vs. heteropolysaccharides

• Homo - consist of one monomer unit

• Hetero - consist of multiple different monomer units

Linear vs. branched polysaccharides

• Linear - contain one type of glycosidic bond

• Branched - contain multiple different types of glycosidic bonds

Glycogen (structure, function)

• Homopolymer of glucose

• Contains mainly alpha1→4 glycosidic linkages

• Branching with alpha1→6, every 8-12 residues

• Acts as a storage polysaccharide in animals, water insoluble, highly branched

• Contains many non-reducing ends for rapid phosphorylation (breaks down glycogen) so glucose can be mobilized

Starch

• Homopolymer of glucose

• Made up of two polysaccharides:

  • Amlyose - contains alpha1→4 glycosidic linkages, linear

  • Amylopectin - contains branching with alpha1→6 every 24-30 residues

• Acts as storage polysaccharide in plants, water insoluble, moderately branched

Cellulose (structure, function)

• Homopolymer of glucose

• Contains beta1→4 glycosidic linkages

• Linear

• Hydrogen bonding occurs between adjacent monomers and chains - forms stable, dense sheets

• Most abundant polysaccharide in nature (plant cell walls), water insoluble, cannot be digested by humans

How do linkages affect structure in polysaccharides?

Alpha glycosidic linkages between monomers introduces a slight kink in the chain → leads to helical polymers

Starch detection

• Starch forms long helices than bind polyiodine

• Transfer of electrons from starch to iodine allows the complex to absorb yellow-red light (appears as blue color)

Major pathways of glucose utilization

• Extracellular matrix/cell wall polysaccharides (e.g. cellulose)

• Storage (e.g. glycogen, starch)

• Oxidation via glycolysis (convert to pyruvate)

• Oxidation via pentose phosphate pathway (convert to ribose-5-phosphate)

Glycolysis (overall purpose, outcome)

• Glycolysis is a series of enzyme catalyzed reactions in which glucose is converted to pyruvate

• Pyruvate can be further oxidized in the citric acid cycle or serve as a precursor in biosynthesis

Glycolysis (overall reaction mechanism)

Breakage of the C3-C4 bond in one D-glucose to produce 2 net ATPs and 2 pyruvate molecules

Glycolysis Step 1

• Phosphorylation of glucose

• Substrate: glucose, Product: glucose-6-phosphate

• Enzyme: hexokinase, Cofactor: Mg2+

  • Mg2+ binds ATP to shield its negative charges

• Negative delta G (very favorable, irreversible)

• Regulated by substrate inhibition (excess substrate binds allosterically to enzyme)

Hexokinase I

• Found in muscle, brain

• Ensures that cells receive enough glucose for basic energy needs, regardless of fluctuations in glucose levels

• Has a low Ks (high affinity for glucose)

• Inhibited by G-6-P

• Plot vi vs. [glucose] → sigmoidal binding curve

Hexokinase IV

• Found in liver

• Clears excess glucose from the blood for storage as glycogen

• Very high Ks (low affinity for glucose)

• High blood glucose → transported into cell → G-6-P → F-6-P → promotes release of kinase from nucleus to cytoplasm → increased production of G-6-P

  • If F-6-P builds up, hexokinase release is inhibited (acts as signal that liver is well-stocked with energy)

• Plot vi vs. [glucose] → positive cooperativity

Glucose-6-Phosphatase

• Liver enzyme

• Removes phosphate group from G-6-P to release glucose into bloodstream

Glycolysis Step 2

• Phosphohexose isomerization

• Substrate: G-6-P, Product: F-6-P

• Enzyme: phosphoglucoisomerase, Cofactor: Mg2+

• Postive delta G (reversible)

  • Paired with favorable next step to drive reaction forward

• Regulated by substrate inhibition (excess substrate binds allosterically to enzyme)

Glycolysis Step 2: intermediate

• Enediol

• Formed through acid-base catalysis

Glycolysis Step 3

• 2nd priming phosphorylation

• First committed step of glycolysis

• Substrate: F-6-P, Product: frustose 1,6-biphosphate

• Enzyme: PFK-1 (phosphofructokinase-1), Cofactor: Mg2+

• Negative delta G (favorable, irreversible)

Phosphofructokinase-1 regulation

• Positively regulated by ADP, AMP

  • Low energy in the cell promotes glycolysis

• Negatively regulated by ATP, citrate

  • High energy in the cell/bottleneck in TCA inhibits glycolysis

• Plot enzyme activity vs [F-6-P]

  • Low [ATP] → high substrate affinity

  • High [ATP] → low substrate affinity

Glycolysis Step 4

• Aldol cleavage of F-1,6-bP

• Substrate: fructose 1,6-bisphosphate, Products: glyceraldehyde 3-phosphate, dihydroxyacetone phosphate

• Enzyme: aldolase

• Positive delta G (standard conditions don’t exist in cell, reaction driven by product consumption)

Mechanism of class I aldolases

• Covalent catalysis

• Intermediate = protonated Schiff’s base

Glycolysis Step 5

• Triose phosphate interconversion

• Substrate: dihydroxyacetone phosphate, Product: glyceraldehyde 3-phosphate

• Enzyme: triose phosphate isomerase

• Positive delta G (unfavorable/reversible, keep GAP concentration low to drive reaction forward)

• DHAP must be converted to GAP to proceed with next phase of glycolysis

Overall product of glycolysis preparatory phase

• Lysis of C3-C4 bond in D-glucose yields 2 D-glyceraldehyde-3-P

• Costs 2 ATP

Glycolysis Step 6

• Oxidation of GAP (oxidation-reduction reaction)

• First energy yielding step in glycolysis

• Substrate: glyceraldehyde 3-phosphate, Product: 1,3-biphosphoglycerate

• Also, oxidation of aldehyde with NAD+ gives NADH

• Enzyme: glyceraldehyde 3-phosphate dehydrogenase

• Postive delta G (coupled with next reaction to drive forward)

Glyceraldehyde-3-Phosphate catalysis

• Performs covalent catalysis

• Has cysteine residue in active site

  • Produces thiohemiacetal and thiohester intermediates

Glyceraldehyde-3-Phosphate inhibition

• Iodoacetate irreversibly inhibits GAPDH by covalently modifiying its active site Cys and preventing formation of thiohemiacetal and thiohester intermediates

• Arsenate reacts with the thiohester intermediate (instead of phosphate group), decoupling 1,3-bisphosphoglycerate and ATP synthesis

  • No ATP produced → glycolysis is unfavorable

NAD+ (recognize structure)

• NAD+ cofactor is used in catabolism (breaking down, making ATP)

• NADP+ cofactor is used in anabolism (building up, spending ATP)

  • NADP+ has similar structure as NAD+, 2’ hydroxyl on ribose is esterified with phosphate

Glycolysis Step 7

• 1st production of ATP

• Substrate: 2 1,3 bisphosphoglycerate, 2 ADP, Product: 2 3-phosphoglycerate, 2 ATP

• Enzyme: phosphoglycerate kinase

• Negative delta G (drives aldolase, TPI, and GAPDH reactions forward)

Glycolysis Step 8

• Migration of the phosphate by PGMutase

• Substrate: 3-phosphoglycerate, Product: 2-phosphoglycerate

• Enzyme: phosphoglycerate mutase

• Positive delta G (unfavorable/reversible, reactant concentration kept high to push reaction forward)

PGMutase mechanism

• One active-site histidine is posttranslationally modified to phosphohistidine

• Phosphohistidine donates phosphate to 3-phosphoglycerate at C2, before retrieving another phosphate from C3

Glycolysis Step 9

• Dehydration of 2-PG to PEP (acid-base catalysis)

• Substrate: 2-phosphoglycerate, Product: phosphoenolpyruvate, water

• Enzyme: enolase

• Positive delta G (product concentration kept low to drive reaction forward)

Enolase mechanism

• Acid-base catalysis

• Goes through enolate intermediate (not covalent)

Glycolysis Step 10

• 2nd production of ATP

• Substrate: 2 phosphoenolpyruvate + 2 ADP, Product: 2 pyruvate, 2 ATP

• Enzyme: pyruvate kinase

• Negative delta G