Dr. DerVartanian is ill and will likely not be able to give lectures this week.

Today’s slides will be put on-line today, and are designed to introduce you to glycolysis.

You should use these slides, along with Dr. DerVartanian’s notes, and your reading of chapters 15-17 (and chapter 6, pages 100-103), TO STUDY THE MATERIAL and PREPARE for next week’s exam.

Glucose + 6 O2 6 CO2 + 6 H2O

+ 6

6+ 6

~280

0 kJ

mol

-1C

ombu

stio

n: ∆

G L

ost

∆G stored2NAD++4e-2NADH2ADP 2ATP

Glycolysis

Citric Acid Cycle

∆G stored8NAD++16e-8NADH2GDP(ADP) 2GTP(ATP)2Q + 4e- 2QH2

Glycolysis and Citric Acid Cycle

Glucose = 30-32 total ATPs during aerobic metabolism.

hexose (6C) stage: 2 ATP’s consumed.triose 2(3C) stage: 4 ATP’s produced.

Net: 2 ATP’s.ALSO, 2 NADH Moleculesand Pyruvate

Hex

ose

Trio

se

Net Reaction:Glucose + 2 ADP + 2 NAD+ + 2 Pi

2 Pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O

Pyruvate catabolism produces most of the energy in mammalian cell!

8NAD++16e-8NADH2GDP(ADP) 2GTP(ATP)2Q + 4e- 2QH2

Energy carriers (ATP/NADH) are in

all life forms

ATP EquivalentsATP = 1NADH = 2.5 (1.5)*QH2 = 1.5GTP = 1

*It costs energy to transport NADH electrons into the mitochondria of some cells

So, glucose = 30-32 ATP’s!

Glycolysis and Citric Acid Cycle

Consume energy to setup Stage 2

Trap Glc in cell

Essential Substrate for

Stage 2

Glycolysis: Step 1, HexokinaseC6

Hexokinase drives passive transport of Glucose

Glc

Glc Glc-6-Phexokinase

glucokinase

‘Passive’ Glc transporters in membrane

Product Inhibition

Metabolically irreversible rxn. It is inhibited by the G-6P productANDSubstrate Availability

Recall: hexokinase uses induced fit

Glycolysis: Step 2Glucose 6-Phosphate Isomerase

Stereospecific: uses -D-Glc-6P; produces -D-fructose-6P

Aldose Ketose

Ultimate cleavage site

Stereospecific: ring opening leads to ~30 % Beta G-6P, what drives the Beta to Alpha?

Aldose to ketose conversion

Step 3, Phosphofructokinase-1 PFK-1

Metabolically irreversible rxn under cellular conditions. It is an allosteric enzyme and a REGULATORY CONTROL step for glycolysis (ATP, AMP and citrate). (see page 299)

First COMMITTED step of glycolysis

ALSO Fructose-2,6-bisphosphate (F-2,6-BP)!!!!!!

Glycolysis: Step 4, Aldolase

Reaction is very Unfavorable (∆G°= 5.7 kcal mol-1)Rapid depletion of 2 products in subsequent steps drives rxn

Rxn is near equilibrium

Keq 1

10, 000

ONLY GAP can enter stage 2!.

We must convert DHAP to a

second molecule of GAP!

Step 5, Triose Phosphate Isomerase

near equil.

AldoseKetose

ONLY GAP can enter stage 2!

Stage 2: Energy Production ONLY GAP can enter stage 2!

So far, we have consumed 2 molecules of ATP

Energy Rich Molecules produced in stage 2:NADHATPPyruvate!!

Glycolysis: Step 6Glyceraldehyde 3-Phosphate Dehydrogenase

Higher group transfer potential than ATP

High Energy Mixed

Anhydride

Generates NADH!!!!!Redox reaction?

Adds or removes a double bond OR Oxygen OR Sulfur atom

Redox RXNs: Follow the double bonds or the addition of an Oxygen.

Great Example of how enzymes use coupled reactions to do difficult chemistry: Formation of a high-energy molecule for making ATP!

Mixed anhydride of phosphoric

acid and a carboxylic acid

NADH, the other energy carrier in the cell. NADH oxidation in the mitochondria produces 2.5 ATP molecules

Carrying 2 e’sNAD+ is a cellular oxidant!

Glyceraldehyde 3-Phosphate Dehydrogenase: Coupled RXN

∆G

RXN Coord.

1) oxidation

∆G

RXN Coord.

2) dehydration

∆G

RXN Coord.

Thioester‘covalent catalysis’

Coupled Rxn

Traps the dG from oxidation to drive phosphorylation

1)

2)

E

EO

Thioester‘High Energy’

EsterCompare leaving

groups!

GAP Dehydrogenase

NAD+

3-PG1,3-BPG

Nucleophilic Addition

The thiohemiacetal oxyanion

tautomerizes to produce an

unstable carbanion, which is easily

oxidized.

Thiohemiacetal Oxidation

NADH The favorable energy from the favorable

oxidation is stored in the high energy thioester (yellow

shading). The attacking phosphate

substitutes the thiolate. What is the

thiol pKa?

Nucleophilic Substitution

(GAPDH)

Step 7, Phosphoglycerate Kinase

Near equilibrium rxn. Reversibility is important for reverse step in glucose synthesis (gluconeogenesis).

Substrate level phosphorylation-Nucleotide diphosphate phosphorylatedDonor is not a nucleotide

First ATP generating stepHigh Energy Mixed

Anhydride

Named for the reverse reaction

Glycolysis: Step 8,9 and 10

High Energy phosphate Donor

Phosphoenolpyruvate

Acidic Proton

Enolase will protonate the hydroxyl to form H2O

Hex

ose

Trio

se

If given you the enzyme names. You must know the reactions (be able to draw the sugar substrates and products)

Know GAPDH: example of elegant coupled chemistry (GAPDH).

Aldolase: largest uphill reaction (standard dG). What drives it?

Triose phosphate isomerase. What drives it

All energy producing and consuming steps

Be able to draw the sugars if given a name. Know the names

What happens if there is no NAD+

But No ATP input!!

Anaerobic Redox Balancing

If a cell is not able to regenerate NAD+ needed by GAPDH, glycolysis will stop!

Net Reaction:Glucose + 2 ADP + 2 NAD+ + 2 Pi

2 Pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O

Lactate

Getting rid of Electrons!

Redox Balancing

Anaerobic‘Yeast’

Anaerobic‘Oxygen starved Muscles’ Aerobic Oxidative

PhosphorylationNADHNAD+

Getting rid of Electrons!

The Most efficient use of glucose!NEEDS a mitochondria!!

Alanine (Cahill) Cycle- pyruvate produced in muscle cannot be exported. In the Cahill Cycle, the amine groups of AA’s are transferred to pyruvate to produce alanine. The alanine is exported from the muscle to the liver, where deamination produces pyruvate for gluconeogenesis.

Cori cycle

RBCs are also a major source of lactate (why?).

Gluconeogenesis: The Cori and Alanine (Cahill) Cycles (Liver).The Cori Cycle converts Lactateproduced in muscle during anaerobic respiration to glucose in the liver to control blood sugar.

Also RBCs!

During starvation, muscle protein catabolism produces energy and pyruvate. Pyruvate can be used to make OAA for Glucose and FAsynthesis in the Liver via the Cahill Cycle.

** *

1. Hexokinase3. Phosphofructokinase 110. Pyruvate Kinase

Three Metabolically Irreversible Reactions

Most reactions are near equilibrium in the cell, and have G close to zero

The table legend is wrong! Small +dG is NO PROBLEM as long as a larger –dG is downstream!

Will the pathway work based on the standard dG?

Glucose-6-phosphataseHexokinase

Allosteric Control of Glycolysis and Gluconeogenesis

We will learn about the Insulin and Glucagon response at the end of gluconeogenesis

Glc

Glc Glc-6-Phexokinase(glucokinase)

‘Passive’ Low affinity Glut 1 transporters in membrane

PFK-1F6P

Insulin regulated High affinity Glut4 transporters. Glut4 inserts in membrane in response to insulin to increase Glc transport

Regulation1) Feedback Inhibition

by product2) Substrate Availability

F-1,6-BP

PGI

Regulation of Hexokinase

Why is Hexokinase NOT a major control point in glycolysis?

Glycolysis

Product Inhibition

What drives Glc import?

See Table 16.3

Glucose-6-phosphataseHexokinase

Allosteric Control of Glycolysis and Gluconeogenesis

Phosphofructokinase is the most important control site in mammalian glycolytic pathway.

Enzyme is 340-kDa tetramer

Allosteric Regulation of PFK-1 Activators*AMP- says ATP low, make more *F-2,6-BP*- says blood Glc High

Inhibitors*ATP**- ATP stores are high

*Citrate- CAC is stopped!*H+- Too much lactic acid!

Allosteric Site

Active Site

*F-2,6-BP is NOT F-1,6-BP!WE WILL LEARN ABOUT THE IMPORTANT ROLE OF F-2,6-BP WHEN WE TALK ABOUT GLUCONEOGENESIS!

*

Does it make sense that ATP is an allosteric inhibitor?

Look at the ‘0’ F-2,6-BP ATP saturation curve. Why are Low concentrations of ATPrequired for PFK-1 to function? Why do High concentrations of ATPinhibit?Notice that the activator F-2,6-Bp can negate the inhibitory effect of high ATP.

High [AMP]

ATP is also substrate in the reaction! Allosteric Site

Active Site

Fructose-2,6-biphosphate Allosteric Regulation of PFK-1 with AMP/ATP

Glucose-6-phosphataseHexokinase

Allosteric Control of Glycolysis and Gluconeogenesis

Allosteric and Covalent Regulation of Pyruvate Kinase*

Allosteric ActivatorsF-1,6-BP: says PFK-1 working

Allosteric InhibitorsATP- ATP stores are highAlanine- From the Cori Cycle/

gluconeogenesis is running

*Named for the reverse reaction

PKA PP1

INHIBIT: When blood [Glc] drops, glucagon signals protein kinase A (PKA) to phosphorylate pyruvate kinase (Liver only)

ACTIVATE: When Blood sugar is high, Insulin signals phosphoprotein phosphatase 1 (PP1) to dephosphorylate pyruvate kinase.

WE WILL DISCUSS THE HORMONAL REGULATION OF PK at the END OF Gluconeogenesis!

Coordinated Regulation of glycolysis in muscle

PFK-1: Key regulatory enzyme in glycolysis.We will learn more in gluconeogenesis!

Contingent on glycogen levels!