BIOCH 200: Slide 9 Glycolysis + Pyruvate Dehydrogenase (PDH)

Is glycolysis catabolic or anabolic?

Catabolic

What is the overall purpose of Glycolysis?

1 Glucose —> 2 Pyruvate

Does substrate level Phosphorylation occur in glycolysis?

Yes

• Generates ATP directly

How does Glycolysis generate NADH?

From the OXIDATION of metabolites (catabolic)

Where does Glycolysis Occur?

Cytoplasm of the cell

• Outside of the mitochondria

Gluconeogenesis? Glycogenolysis? Glycogen synthesis?

At which step of glycolysis does Glycogen synthesis/glycogenolysis tie in?

G6P Enters Glycolysis

What are the 2 stages of Glycolysis?

1. Energy investment

   • ATP = used

2. Energy payout

   • ATP = made

   • NADH = made

Which steps of glycolysis = energy investment and which are payout

All steps with HEXOSE (6 C) = Invest

All steps with TRIOSE (3C) = Pay out

What are the 4 enzymes to know for glycolysis?

Associated with:

• 2/2 rxns that use ATP

• 2/3 E capture steps (6=NADH + 10 = ATP)

Which 3 enzymes of Glycolysis are regulated?

• Hexokinase

• Phosphofructokinase-1 (PFK-1)

• Pyruvate kinase

What is a commonality of the rxns that have regulated enzymes?

They are IRREVERSIBLE

In which Rxns is ATP used?

STEP 1: Glucose —> G6P (enzyme: Hexokinase

STEP 3: F6P —> Fructose-1,6-BP (Enzyme: PFK-1)

What is the commonality between the 2 steps that require ATP investment as E?

They are IRREVERSIBLE + catalyzed by regulated enzymes

What is the Net RXN of the E investment Phase?

Glucose + 2 ATP —> 2 GAP + 2 ADP + 2H+

What kind of rxn is step 1: Glucose —> G6P?

Phosphate transfer

• Hexokinase = phosphorylate

What kind of rxn is STEP 2: G6P —> F6P

Isomerization

• Glucose + fructose are isomers

What kind of rxn is STEP 3: F6P —> F-1,6-BP?

Phosphate transfer rxn

• Phosphofructokinase-1 = Phosphorylation

A. - 46 KJ/mol

B. - 22 KJ/mol

C. - 18 KJ/mol

D. 18 KJ/mol

E. 46 KJ/mol

C. Net = -32 + 14 = -18

What kind of rxn is STEP 4: F-1,6-BP —> DHAP + GAP?

LYSIS of glycolysis

What is the relationship between DHAP + GAP?

They are ISOMERS

Why is there 2 pyruvates per 1 Glucose?

DHAP + GAP = Isomers

• DHAP = converted to GAP = 2 GAP

• Every rxn from GAP —> Pyruvate = occurs twice

What is the net rxn of the E payout steps?

2x: GAP + NAD+ + Pi + 2ADP —> Pyruvate + NADH + 2 ATP + H2O + H+

What kind of rxn is STEP 6: GAP/G3P —> 1,3- BPGlycerate?

The OXIDATION part of glycolysis

• PHOSPHATE ADDITION (Pi)

• E capture step (NADH)

What type of Rxn is STEP 7: 1,3-BPG —> 3-PG

Substrate Lvl Phosphorylation (aka. Phosphate-transfer)

• E capture step (ATP captured directly as result of substrate lvl phosphorylation)

• 1,3-BPG = has large phosphate transfer potential

Why does 1,3-BPG have a large phosphate transfer potential

It has an Acyl Phosphate

• the product it forms is resonance stabilized

True or False: ALL substrate lvl phosphorylation steps = E capture steps?

True

• but not all E capture steps = substrate lvl phosphorylation

What kind of Rxn is STEP 8: 3-PG —> 2-PG

ISOMERIZATION

What kind of Rxn is STEP 9: 2PG —> Phosphoenolpyruvate (PEP)?

DEHYDRATION

• Double bond = generated

What kind of rxn is STEP 10 (last step): PEP —> Pyruvate?

Substrate Lvl Phosphorylation (Phosphate- transfer)

• E capture step (ATP directly from substrate lvl phosphorylation)

True or False: Normally the Rxn from PEP —> Pyruvate is Reversible but in physiological conditions the reverse rxn is NOT favored?

TRUE

What is Enol pyruvate?

High E molecule

Tautomerizes into Pyruvate

What is the result of the tautomerization of Enol Pyruvate?

Large amount of E = released = MAKES the STEP /rxn IRREVERSIBLE

What is the Overall Balanced Rxn of Glycolysis?

Glucose + 2ADP + 2NAD+ + 2Pi —> 2 Pyruvate + 2 ATP + 2 NADH + 2H+ + 2 H2O

What is the NET ATP yield?

2 ATP

• 2 ATP invested + 4 ATP out

• 4-2 = 2

Which steps are the E capture steps of Glycolysis?

• 6, 7 + 10

  • 7 + 10 = ATP

  • 6 = NADH

Which steps are substrate lvl phosphorylation steps

7 + 10 (generated ATP directly)

Which steps are Phosphate transfer steps?

1, 3, 7 + 10

• All steps where ATP was involved

  • ATP investment

  • Substrate lvl phosphorylation and/or rxns with kinase enzymes

What are the High E molecules involved in Glycolysis

Products: ATP + NADH

Intermediates: Phosphoenolpyruvate (PEP) + 1,3 BPG

• Honorable mention Enolpyruvate + G6P

What are the coupled rxns in Glycolysis

Where ATP = involved

• Invest: 1 + 3

• Payout: 7 +10

What is the net gain when 1 mol of Dihydroxyacetone phosphate (DHAP) is converted to pyruvate?

D. Only go through second phase of glycolysis once

Which steps have a Delta G close to 0 and which have a very negative Delta G?

Close to 0 = REVERSIBLE STEPS

Very negative = IRREVERSIBLE STEPS

What are the Isomerization steps of Glycolysis?

2, 5 + 8

What is the Rate limiting step/ sets pace of Glycolysis?

Step 3: F6P —> F-1,6-BP

Why isn’t step 1 the rate limiting step despite being regulated?

G6P goes into Glycogen synthesis thus the regulation doesn’t control glycolysis exclusively

• Step 3 on the other hand is exclusive to glycolysis

Which step is the oxidation step of glycolysis?

First step of Phase 2

• G3P/GAP —> 1,3-BPG

• NADH = FORMED

Why is Phosphoenolpyruvate a high E intermediate

Enol = unstable, the conversion of PEP —> Pyruvate = highly favorable

• phosphate group also = strained position

What are the 3 regulated steps of Glycolysis + which enzymes regulate them

3 Irreversible steps (1, 3 + 10)

STEP 1: Hexokinase

STEP 3: PFK-1

STEP 10: Pyruvate Kinase

What is the balanced equation for STEP 1?

What is the balanced equation for STEP 3?

What is the balanced equation for STEP 10?

Why is glycolysis regulated (3 reasons)

1. Ensure E needs are met

2. Not wasting Glucose when ATP = Abundant

3. Intermediates may be used in other processes

What 4 major processes regulate the Rate of Flux through metabolic pathways?

1. Substrate availability

2. Alteration of enzyme activity

3. Alteration of amount of enzyme

4. Compartmentation

Define compartmentation

Putting things into storage and Controlling movement of the stored things

What are the 2 types of regulation in glycolysis?

1. Substrate availability

   • Glucose import (transporters)

2. Enzyme regulation (Hexokinase, PFK-1 (rate limiting), Pyruvate Kinase)

Which of the following glycolytic enzymes do expect to not be regulated

D.

What type of Regulation is Hexokinase under?

Product inhibition

• G6P = inhibitor

What is G6P to Hexokinase?

Negative allosteric effector

How is PFK-1 Regulated

Allosterically by:

• PEP

• ADP/AMP

What is PEP (Phosphoenolpyruvates) relation to PFK-1?

Hetero-allosteric Inhibitor/ negative effector/ feed back inhibitor

What do Elevated lvls of PEP indicate

That the products of Glycolysis (Pyruvate) are not being consumed

What is AMP/ADPs relation to PFK-1 allosterically?

Heteroallosteric ACTIVATOR/ Positive effector

Other Allosteric effectors of PEP?

• Fructose-2.6-BP (under insulin + glucagon control) respond to blood glucose

• Fructose 6 Phosphate = Homoallosteric activator

• ATP = HOMOALLOSTERIC inhibitor

Why is the [AMP/ADP] a good indicator of the need of ATP. Why not [ATP] itself?

[ATP] = relatively stable

[AMP/ADP] = increases a lot when ATP = used

What would a graph of activity vs [F6P] look like for PFK-1 with or without PEP and AMP?

What does the sigmoidal curve of Activity of PFK-1 vs. [F6P] graph mean?

Sigmoidal means F6P = Homoallosteric activator

What type of Regulation is Pyruvate kinase under?

Allosteric Regulation

• ATP

• F-1,6-BP

What is the Relationship between ATP and Pyruvate kinase(PK)?

Product inhibition (kind of, other steps also make ATP)

• ALLOSTERIC Inhibitor

What is the relationship between F-1,6-BP and PK?

F-1,6-BP = ACTIVATE

• FEED forward activation

• Heteroallosteric Activator/ positive effector

Which Enzymes Have feedback inhibition/ feed-forward activation/ product inhibition.

Feed back inhibition = PEP on PFK-1

Feed forward activation = F-1,6-BP on PK

Product Inhibition = ATP on PK (Kinda) + G6P on Hexokinase

Gross yield vs. Net yield

Gross = only profit

Net = Profit - Investments

What would a graph of activity vs [PEP] look like for PK with or without F1,6BP and ATP?

True or False: PEP = Homoallosteric Positive effector of PK?

TRUE

• Curve = sigmoidal for graph of PK activity vs [PEP]

Which of the 3 regulated Enzymes are Inhibited by ATP

PK + PFK-1

What does the Regulation of Pk and PFK-1 both by ATP achieve?

• Synchronous regulation of irreversible rxns

  • Flow In = Flow Out

  • MAINTAIN STEADY STATE

Overview of Glycolysis regulation

Overview of Glycolysis RXNS

Is glycogen synthesis anabolic or catabolic?

Anabolic

What is glycogen synthesized from?

G6P

Why does Glycogen produce a NET yield of 3 ATP from glycolysis?

No ATP = needed to generate G6P from glycogen

• skipping the first step = ATP investment step

• Net = 1 ATP invested + 4 ATP obtained

Which of the following does NOT occur in the rxns between F-1,6-BP + 1,3-BPG

A. While 1,3 BPG becomes phosphorylated during the GAPDH reaction, it is through phosphate addition, not transfer.

Which of the following correctly matches the enzyme with its Inhibitor/activator and type of regulation

E.

What are the 3 fates of Pyruvate?

1. Ethanol

2. Lactate

3. Acetyl-CoA (+Oxidative phosphorylation)

Of the 3 fates, which are /is Aerobic and Which are/is Anaerobic

Aerobic = Acetyl CoA

Anaerobic = Lactate + Ethanol

Why is an anaerobic Fate for Pyruvate necessary?

To reoxidize NADH —> NAD+ for the the OXIDATION rxn in glycolysis under anaerobic conditions

What is the purpose of pyruvate metabolism anaerobic?

NADH need to be reoxidized back to NAD+ for Glycolysis to continue

• Oxidative phosphorylation (aerobic)

• Pyruvate reduction (Anaerobic)

  • Ethanol + Lactate

What is the equation which describes the fate of pyruvate in anaerobic muscle.

True or False: Lactate is an acid

FALSE: it is not an acid

True or False: Lactate is a dead end product for all parts of the body.

False

• Muscle = DEAD END product

• Heart =/= Dead end product

Why is lactate considered a dead end product for skeletal Muscle in ANAEROBIC acitivity

We can’t do much with it other than turn it back into Pyruvate

What happens to Lactate in muscle other than staying there?

It gets exported from the muscle in to BLOOD

How is Lactate exported into blood?

Specific membrane transporter proteins

• Symporter with H+

What is the metabolic effect of the export of lactate —> blood? Think Blood, Heart + glycolysis?

• H+ is transported out with lactate = lowers the pH of blood = decrease affinity of Hb for O2 = release of O2 (Bohr effect)

• Lactate in blood can be transported to heart for use

• Prevents the build up of Lactate = prevent build up of pyruvate which would inhibit PK action

What is the ACTUAL source of acid in muscle if Lactic acid is a myth?

Why is lactate not a Dead end product for the Heart?

The heart is ALWAYS under AEROBIC conditions in which lactate can be used as metabolic fuel

How lactate is used as metabolic fuel under aerobic conditions?

What is the biochemical purpose for the production of lactate in muscle cells

A.

What is the Pyruvate dehydrogenase rxn?

It is the aerobic fate of pyruvate

• Pyruvate —> Acetyl CoA

• Links glycolysis to CAC

Where does the pyruvate dehydrogenase rxn occur?

Mitochondrial matrix

What catalyzes the Pyruvate Dehydrogenase rxn?

Pyruvate dehydrogenase Complex

• PDH, PDC