EXAM 2: Part 1 - Glycolysis

cytosolic enzyme

catalyzes glycolysis

Pyruvate

endpoint of glycolysis

Aerobic

Pyruvate converted to CO2 and H2O

Anaerobic

Pyruvate to lactate or ethanol

Pyruvate structure

2C molecule

How much pyruvate is produced?

2/glucose

How does glucose enter

GLUT-1 transporters

What traps glucose in cell

phosphorylation

How much ATP consumed

2, then cleaved into 2 3C molecules

total ATP generated

4

Net ATP

2

E-Conversion Pathway stage 1

No ATP
2 substages: Investment, Splitting

E-Conversion Pathway stage 2

ATP is harvested
Aka ATP yield stage

1a. Investment stage

first stage of E-conversion pathway
Glucose is taken to cell by GLUT-1 transporters/is trapped
Hexokinase phosphorylates glucose
Rxn is unfavorable so 1 ATP invested

Hexokinase

Phosphorylates glucose in 1st part of investment stage

Fructose-6-Phosphate / F-6-P

2nd step in investment stage
1 ATP investment required to create next intermediate → fructose 1,6-biphosphate (F1,6-BP) by PFK-1

Phosphofructokinase / PFK-1

Catalyzes rxn to create fructose 1,6-biphosphate (F1,6-BP) in 1st part of investment stage

Primary site of regulation of glycolysis
Controls flux F-6-P to F-1,6-BP and indirectly, the levels of G-6-P and therefore, inhibition of hexokinase
*** Strongly inhibited by ambient ATP

Splitting Stage

2nd part of E-conversion path
F-1,6-BP is cleaved

F-1,6-BP is cleaved into…

Dihydroxyacetone phosphate (DHAP)
Glyceraldehyde 3-phosphate (GAP)

GAP

Glyceraldehyde 3-phosphate is on direct pathways of glycolysis

Triose phosphate isomerase

readily interconverts isomers to not waste 3Carbon fragments

The Yield Stage

2nd stage of glycolysis
4 molecules of ATP created (net=2)

Substrate-level phosphorylation

process where a high E phosphate compound transfers a phosphate
ADP→ATP

X~P + ADP →

X + ATP

Glyceraldehyde-3-phosphate (GAPDH)

Oxidizes GAP to carboxylic acid → creates 1,3-bisphosphoglycerate (1-3,BPG) (a high E phosphate)
This is an oxred. rxn so NAD+ is reduced to NADH

NADH

e- transporting molecule

Substrate-level phosphorlation

Produces ATP from another high E phosphate compound
PEP now has a high E phosphoryl group and is a substrate for the next highly regulation rcn catalyzed by pyruvate kinase

Pyruvate kinase (PK)

3rd highly regulated step in glycolysis
Utilizes high E bond from PEP to phosphorylate ADP into ATP

How many path does pyruvate have?

3

Possible fates of pyruvate

Ethanol, Lactate, CO2/H2O

No O2 present outcome of pyruvate

ethanol, lactate

Yes O2 present outcome of pyruvate

CO2, H2O

Lactate dehydrogenase / LDH

Regenerates NAD+ consumed in GAPDH
Produces Lactate
anaerobic glyc.
In prok and euk cells

How is lactate formed?

lactic acid formation / anaerobic glycolysis
Pyruvate accepts e- from NADH bc of LDH
No net oxidation-reduction
Regeneration fo NAD+ in the reduction of pyruvate to lactate sustains the continued process of glycolysis under anaerobic conditions

Fermentation

General term for anaerobic metabolism of glucose
Begins with decarboylation of pyruvate bc of pyruvate dehydrogenase
→ acetaldehyde → releases CO2 → NADH is then oxidized by NAD+ → ethanol formation

Do mammalian cells ferment?

NO

Ethanol

Formed by pyruvate by yeast and other microorganisms
Glucose → alcohol = alcoholic fermentation
Process regenerates NAD+ so cycle can continue
NOT in human cells

Glycolysis regulated at 3 kinase rxns

Hexokinase, Pyruvate kinase, Phosphfructose-1

Common characteristics of regulatory enzymes

Allosterically regulated, present at low Vmax in comparison to other enzymes in pathway
Catalyze irreversible rxns

Hexokinase (Muscle)

Inhibited by product - glucose 6-phosphate
High glucose 6-phosphate = low hexokinase activity

When high E charge →

PFK-1 inhibited (steady-state glycolysis)

When E charge low (high AMP) →

PFK-1 active (increased need of glycolysis)

Phosphofructkinase (Muscle)

*Most important control site in the mammalian glycolytic pathway*
High levels of ATP allosterically inhibit enzyme
AMP reverses inhibitory action of ATP
AMP competes with ATP for regulatory binding site but when bound, AMP does NOT inhibit enzyme
AMP is signal for a low E state

Pyruvate Kinase (PK)

Regulated by hexokinase and PFK-1 activity
Allosterically activated by F-1,6-BP (product from PFK-1 rxn) = feed forward regulation

Pyruvate kinase (muscle)

ATP allosterically inhibits
Fructose 1,6-bisphosphate (preceding irreversible step in glycolysis) → activates kinase

Regulation of glycolysis in liver

Liver is biochemically versatile
Liver maintain blood-glucose [_]
Uses glucose to generate reducing power for biosynthesis

High glucose

Glucose stored as glycogen

When low blood-glucose

Liver releases glucose

Glucokinase

An isozyme of hexokinase
Responsible for phosphorylating glucose
Only phosph. glucose when glucose is abundant
Provide G6-P for synthesis of glycogen an for Fatty Acid formation

Isozyme

Enzymes located on different genes/different a.a sequences but catalyze the same rxn

High Km of glucokinase for glucose in Liver

Allow brain and muscle have first dibs on glucose

Insulin

Signals the need to remove glucose from the blood for storage as glycogen or conversion into fat

Phosphofructokinase in Liver

Regulated by ATP - but regulation is not as impt bc liver does not exp sudden ATP needs like brain/musc.
Inhibited by citrate (early intermediate in Citric acid cycle (TCA)

Glycolysis in Liver →

Carbon skeletons for biosynthesis

Fructose 2,6-biphosphate is a key molecules for

…Liver response to changes in blood glucose → overcome ATP inhibit effects and keeps PFK going
As blood glucose increases, fructose 6-phosphate increases in liver

Fructose 2,6-biphosphate abundance

accelerates synthesis of F2, 6-BP

F2, 6-BP

Stimulates glycolysis by increasing PFK’s affinity for fructose 6-phosphate and diminishing the inhibitory effects of ATP → feedforward stimulation

Activation of PFK-1 by F-2,6-BP

PFK is active at low [substrate] in presence of F2,6-BP
ATP, as a substrate initially stimulates rxns, but as [ATP] increases, it acts as an allosteric inhibitor

What form of PK predominates in each organ?

L form in Liver
M in muscle and brain
Phosphorylation of PK occurs in low blood-glucose states
Prevents liver form consuming glucose when it is needed in brain/muscle