Unit 8: Glycolysis, Gluconeogenesis, & Pentose Phosphate Pathway

What all can glucose be converted to?

• Glycogen (storage form)

• Pyruvate (through glycolysis)- NADH is formed & can be cashed in for ATP

  • ATP is formed directly

• Extracellular matrix & complex polysaccharides (used to synthesize structural glycopeptides)

• Ribose 5-phosphate (through Pentose Phosphate Pathway)- required for DNA & RNA & coenzyme A, etc.

  • Produces NADPH

How many steps is glycolysis & what does it convert glucose into?

10 steps & into pyruvate w/ concomitant production of ATP

What is glycolysis the prelude to in aerobic organisms?

Citric acid cycle & oxidative phosphorylation (which together provide most of the energy contained in a molecule of glucose)

What is the dual role of the glycolysis pathway?

• Degrades glucose to generate ATP & NADH (which converts to ATP)

• Provides building block for synthetic reactions

How many ATP per glucose molecule?

32

How many ATP per NAD+ molecule?

2.5

What is the pathway overall?

Glucose → 2x pyruvate → 2 ethanol + 2 Acetyl CoA (can convert to fatty acids of storage OR CO₂ + ATP via citric acid cycle) + 2 lactate

What does conversion to lactate occur in?

“Temporary” anaerobic condition “dead end”

What are the key structures of glycolysis?

• Glucose & fructose (hexoses)

• Dihydroxyacetone, glyceraldehyde, glycerate, & pyruvate (trioses)

• Glyceraldehyde 3-phosphate

• 1,3-bisphosphoglycerate

• ATP

What will all intermediates be in glycolysis (at OH or a CO₂-)?

Phosphorylated

Is the bond involved in glyceraldehyde 3-phosphate a high energy bond?

No! Phosphate group at a -OH group

Is the bond involved in 1,3-bisphosphoglycerate a high energy bond?

Yes! At the carboxylic acid, it is an acid anhydride which is high energy bond similar to ATP

What is oxidation?

Loss of elections, a gain of oxygen, &/or a loss of hydrogen

What is reduction?

Gain of electrons, a loss of oxygen, &/or a gain of hydrogen

What can oxidation not occur w/o?

Reduction (& vice-versa)

What is the phosphoryl transfer?

• A phosphate is transferred to a biomolecule from ATP or vice-versa

• Energy in the form of ATP is either used or formed

• R-OH + ATP → R-(P) + ADP + H+

What is the phosphoryl shift?

• Movement within the same molecule

• No energy is used

What is isomerization (keto-enol tautomerization)?

Aldose → ene-diol → ketose (& vice-versa)

What is dehydration?

• Reversible loss or gain of H₂O

• Normally, this is specific (i.e. only one isomer will be formed)

What is aldol cleavage & condensation (breaks or forms a C-C bond)?

Aldose/ketose + aldose → bigger aldose/ketose

What is the preparatory phase of glycolysis?

• Phosphorylation of glucose & its conversion to glyceraldehyde 3-phosphate

• Costs: glucose & 2 ATP

• Gains: 2 glyceraldehyde 3-P (Gly 3-P)

What is step 1 of glycolysis (hexokinase)?

• 1 of 3 “irreversible” reactions

• Requires a kinase enzyme

• Glucose → glucose 6-phosphate

• Glucose enters cell via a specific transporter (GLUT → multiple isomerases)

• Traps glucose in cell

What is step 2 of glycolysis (phosphohexose isomerase)?

• Catalyzes the reversible isomerizaiton of an aldose (glucose) to a ketose (fructose)

• Glucose 6-phosphate (hemiacetal) → fructose 6-phosphate (hemiketal)

• Sugars need to revert back to their linear forms, isomerization via ene-diol, recycling

• Allows C-OH group to be available

• Phosphohexose isomerase is used

What is step 3 of glycolysis (phosphofructokinase-1 (PFK-1))?

• The 1 refers to the position of phosphorylation

• This is the first committed step in glycolysis (b/c major site of control)

• Essentially irreversible

• Total cost after first 3 steps is 2 ATP

• Fructose 6-phosphate → fructose 1,6-biphosphate

• Phosphofructokinase-1 is used

What are compounds w/ 2 phosphate groups at 2 different positions known as?

Biphosphates

What are compounds w/ 2 phosphates linked together are known as?

Diphosphates

What is step 4 of glycolysis (aldolase)?

• Catalyzes the reversible cleavage of a hexose to 2 trioses

• Reaction occurs after F-1,6-BP is converted to its linear form

• Fructose 1,6-bisphosphate → dihydroxyacetone phosphate + glyceraldehyde 3-phosphate

• Aldolase is needed

• Even though large pos delta G, reversible at physiological concentrations

What is step 5 of glycolysis (triose phosphate isomerase)?

• Mechanism is identical to phosphohexose isomerase (step 2)

• Normal equilibrium here is tilted strongly (>90%) towards the formation of dihydroxyacetone phosphate

  • Tilts towards glyceraldehyde 3-P by removal of Gly-3-P via subsequent steps

• Dihydroxyacetone phosphate → glyceraldehyde 3-phosphate (move forward; 2 molecule of Gly-3-P)

• Triose phosphate isomerase is needed

What is the summary of the preparatory phase of glycolysis?

• One 6-carbon molecule is converted to two 3-carbon molecule

• 2 ATP have been used

• Pathway proceeds w/ glyceraldehyde 3-phosphate

• Contains committed step & 2 irreversible reactions (Control)

• All energy will be formed from the 2 Gly-3-P

How many ATP per Gly-3-P?

17

What is the overview of the phase 2: payoff phase?

• Oxidative conversion of glyceraldehyde 3-phosphate to pyruvate & coupled formation of ATP & NADH

• Cost entering the payoff phase w/ 2 ATP

• Produced: 2 NADH, 4 ATP, 2 pyruvate

• Net: 2 NADH, 2 ATP, 2 pyruvate

How many ATP per pyruvate?

12.5

What is step 6 of glycolysis (glyceraldehyde 3-phosphate dehydrogenase)?

• Aldehyde of glyceraldehyde 3-P is oxidized to an acid anhydride

• NAD+ is reduced to NADH

  • NADH can be “cashed in” later for energy

• Dehydrogenase enzymes are involved in ox/red reactions

• Glyceraldehyde 3-phosphate + inorganic phosphate → 1,3-bisphosphoglycerate

• Glyceraldehyde 3-phosphate dehydrogenase is needed

• Dehydrogenase means “loss of hydrogen”

What is the binding site of the glyceraldehyde 3-P dehydrogenase?

Cysteine

What happens in the mechanism of the last 5 steps of glycolysis?

• 1: Formation of enzyme-substrate complex (active site Cys)

• 2: Nucleophilic cys reactions w/ the aldehyde (linkage between substrate & -S- group of Cys)

• 3: Enzyme-substrate intermediate is oxidized by NAD+ bound to active site (NADH is reduced & other is oxidized to a thio-ester)

• 4: NADH product leaves the active site & replaced by NAD+ (Phosphate displaces Cys-SH & regenerates the enzyme)

• 5: Linkage between substrate & enzyme undergoes phosphorolysis, releasing the second product

What is step 7 of glycolysis (phosphoglycerate kinase)?

• Enzyme is named for reverse reaction

• ATP is directly formed from 1,3-bisphospoglycerate (transfer type is known as substrate level phosphorylation)

• Combinations of reactions of 6 & 7 drive this pathway in the forward direction

• Regenerates 2 ATP molecules that were consumed in the preparatory phase

• Distintinguishes this from oxidative phosphorylation (requires O₂ & other pathways)

  • How NADH is converted to ATP

What is step 8 of glycolysis (phosphoglycerate mutate)?

• This step & step 9 are “set-up” steps

• Catalyzes a reversible phosphoryl shift

  • Moves a phosphate within the structure

• 3-Phosphoglycerate → 2,3-phosphoglycerate → 2-Phosphoglycerate

• Phosphoglycerate mutase is needed

• The 2-phosphoglycerate formed is a phosphate ester (low energy)

• Requires a P-His (also body can use Ser for other reactions)

What is step 9 by glycolysis (enolase)?

• Catalyzes a dehydration reaction

• Result of dehydration is the generation of a phosphate group that has high transfer potential

• 2-Phosphoglycerate → Phosphoenolpyruvate

• Enolase is needed (Adds across a bond- reversible)

What is step 10 of glycolysis (pyruvate kinase)?

• Similar to phosphoglycerate kinase (step 7), this is named for reverse reaction

  • Major difference is reaction is essential irreversible

• Generates pyruvate (end product of glycolysis as well as ATP)

• Substrate-level phosphorylation (Produces directly)

• Phosphoenolpyruvate → pyruvate

• Pyruvate kinase is needed

What is the summary of 4 kinases in glycolysis?

• 3 irreversible

• 1 reversible

What is the overall input & output of glycolysis?

• Input: 1 glucose & 2 ATP (prep), 2 NAD+, 4 ATP, 2 Pi (payoff)

• Output: 2 pyruvate, 2 ATP, & 2 NADH (prep), 2 ATP (payoff)

Glucose + 2 Pi + 2 ATP + 2NAD+ → 2 pyruvate + 2 ATP + 2 NADH

What is up with the feeder pathway for glycolysis?

• Entry of galactose (lactose component) & fructose (sucrose component)

• Only a few new types of enzymes

What cleaves the disaccharide sucrose?

Sucrose

What cleaves the disaccharide lactose?

Lactase

What happens with lactose → D-Galactose?

• Epimer at C4 so cannot convert to glucose via ene-diol

• ATP is required → UDP-galactose → UDP-glucose (epimerase needed)

What happens with D-Mannose?

• C2 epimer at glucose

• Change from aldose to ketose removes difference

What happens with the 2nd pathway for fructose?

Same types of enzymes but in a different order

What is the key point for the feeder pathways of glycolysis?

Energy cost for all 4 sugars is the same → 2 ATP

What does the conversion of galactose to glucose require?

Epimerase enzyme & 2 ox/red reactions

What is the enzyme for UDP-galactose → UDP-glucose?

UDP-glucose epimerase

What is the central role of pyruvate?

• Conversion of sugar to amino acid

• Ethanol is formed in yeast & other several microorganisms

• Conversion from glucose to ethanol is fermentation

  • Can occur anaerobically (no net ox/red or no net NAD/NADH₂)

Glucose + 2Pi + 2ADP + 2H+ → 2Ethanol + 2CO₂ + 2ATP + 2H₂O

What is lactate formed in?

Variety of microorganisms & in higher organisms when oxygen concentrations are limited (e.g. in muscle tissue during intense activity)

• NADH (product of step 6) is being formed faster than oxidative phosphorylation can regenerate it

• Conversion allows glycolysis to occur “anaerobically”

Pyruvate → Lactate (“dead end’)

• In aerobic conditions, lactate is converted back to pyruvate

Net: Glucose + 2Pi + 2ADP → 2lactate + 2ATP + 2H₂O

What is the purpose of the formation of lactate in skeletal muscles?

Regenerate NAD+ for glycolysis

Where can lactate be transported?

To liver for gluconeogenesis & returned to muscle (Cori Cycle)

• Muscles function “anaerobically” by converting pyruvate to lactate

• Lactate is released in blood, travels to liver where converted back to glucose & reused by muscle

What is gluconeogenesis?

• Defined as the formation of glucose from noncarbohydrate sources (pyruvate can be formed by other pathways, other intermediates can enter); does use many intermediates of glycolysis

What is gluconeogenesis not a reversal of?

Glycolysis

• B/C there were 3 reactions in glycolysis that are irreversible b/c we need to “bypass” these

• Uses the 7 reversible steps of glycolysis

What is bypass #1 of gluconeogenesis (converting pyruvate to Phosphoenolpyruvate)?

• Requires carboxylate enzyme → adds CO₂-

• Kinase enzyme → adds phosphate

Bicarbonate + pyruvate + ATP → Oxaloacetate → Phosphoenolpyruvate

• Biotin & pyruvate carboxylase to form oxaloacetate

• PEP carboxykinase to form phosphoenolpyruvate

What is the cost of conversion for 1 pyruvate in bypass 1?

2 ATP

What does biotin act as?

Activated carrier of CO₂ (readily reversible HCO₃-)

What happens with the pyruvate in bypass 1?

• Transported from the cytosol into mitochondria prior to being converted to oxaloacetate

  • Oxaloacetate cannot be transported out of the mitochondria. Must be either converted to phosphoenolpyruvate or malate

  • Reversible conversion of oxaloacetate to malate by malate dehydrogenase is also part of citric acid cycle

What are oxaloacetate & malate intermediates for?

Citric acid cycle

Why is the bypass 1 important?

Reversible red/ox assures that there is adequate NADH in the cytosol for gluconeogenesis

What is bypass #2 in gluconeogenesis (converting fructose 1,6-bisphosphate to fructose 6-phosphate)?

• Accomplished by a phosphatase enzyme

• Reaction is highly exergonic & is catalyzed by fructose 1,6-bisphosphatase (FBPase-1)

Fructose 1,6-bisphosphate + H₂O → fructose 6-phosphate + Pi

• Cleaves a low energy bond (phosphate ester)

• No ATP is formed

What type of enzyme adds phosphates?

Kinase

What is bypass #3 for gluconeogenesis (converting glucose 6-phosphate to glucose)?

• Requires specific phosphatase enzyme, glucose 6-phosphatase (only used in gluconeogenesis)

Glucose 6-phosphate + H₂O → Glucose + Pi

• Glucose 6-phosphatase is ONLY found on the luminal side of the endoplasmic reticulum of hepatocytes, renal cells, & epithelial cells

• WHY is this enzyme limited to these cells?

  • These cells are the only ones that share (supply) glucose to the blood & other tissues

What is the overall gain of energy in glycolysis?

2 ATP & 2 NADH

What is the overall cost of energy in gluconeogenesis?

6 ATP & 2 NADH

What is the pentose phosphate pathway?

• Has 2 branches or phases

• Also known as pentose phosphate shunt

• Purpose of pathway is to produce 2 key components:

  • NADPH: “reductive power” → required in biosynthesis reactions

  • Ribose 5-Phosphate (key standard component of all DNA, RNA, ATP, Acetyl CoA, etc.)

What is the oxidative branch/phase?

Glucose 6-phosphate → irreversible (glucose 6-phosphate dehydrogenase & NADP+ into NADPH + H+) → 6-phospho-gluconeogenesis-sigma-lactone → reversible break ring (lactonase(esterase) + H₂O) → 6-phospho-gluconate → irreversible (6-phosphoglycerate dehydrogenase & NADP+ into NADPH + H+ & CO₂) → ribulose 5-phosphate → (phosphopentose isomerase) → ribose 5-phosphate

What is the net reaction of oxidative branch/phase?

Glucose 6-P + 2 NADP+ → Ribose 5-P + CO₂ + 2 NADPH

What is the reaction catalyzed by in the oxidative branch that is irreversible & what does major control depend on?

• Catalyzed by glucose 6-phosphate dehydrogenase (major control site)

• Depends upon: substrate [NADP+], higher concentration signals the enzyme to convert it to NADPH

What if you only needed either NADPH or Ribose 5-Phosphate?

Use the non-oxidative branch

What is the nonoxidative branch/phase?

• Converts ribulose 5-phosphate to fructose 6-phosphate & glyceraldehyde 3-phosphate & vice-versa

• Does not involve ox/red reactions; no change in NAD+/NADH or NADP+/NADPH ratios

• Transketolase & transaldolase needed

What is transketolase?

• Transfers 2 carbon units b/w ketoses & aldoses

• Requires thiamine pyrophosphate

• Readily reversible

What is transaldolase?

• Transfers 3 carbon units

• Does not require thiamine pyrophosphate

• Works through Schiff Base formation w/ a Lys nitrogen

What is the summary of the nonoxidative phase?-

3 Ribulose 5-phosphate → 2 fructose 6-phosphate + glyceraldehyde 3-phosphate

• Relative needs of ribose 5-phosphate, NADPH, pyruvate, & ATP determine flow of glucose 6-phosphate through pentose phosphate shunt

What are the key points about the oxidative vs. nonoxidative branch?

• Oxidative is one-way street (irreversible)

• Nonoxidative is reversible two-way street

When is all energy formed after?

Production of glyceraldehyde 3-phosphate

What does each glyceraldehyde 3-phosphate molecule (3 carbon phosphorylated sugar) produce (in steps 6-10)?

• 1 pyruvate → 12.5 ATP (through citric acid cycle)

• 1 NADH (step 6)→ 2.5 ATP

• 2 ATP (steps 7 & 10)

• Total: 17 ATP/gly-3-P

What pathway would you use if a cell needed ribose 5-phosphate but not NADPH?

Glycolysis & nonoxidative branch of PPP

What pathway would you use if a cell needed ribose 5-phosphate & NADPH?

Glycolysis & oxidative branch of PPP

What pathway would you use if a cell needed NADPH but not ribose 5-phosphate?

Glycolysis, oxidative branch, non-oxidative branch, gluconeogenesis