BIOL 1510 Chapter 14

Fates of glucose

storage molecules, ECM and cell wall polysaccharides, energy synthesis, structural molecules

Amount of energy released per one glucose molecule

~3000 kcal/mol

Two phases of glycolysis

1. Preparatory/energy investment phase

2. Payoff phase

1st step of glycolysis

Hexokinase phosphorylates glucose into glucose 6 phosphate

Purpose of turning glucose into glucose-6-phosphate

Traps the molecule inside the cell and keeps it from diffusing out. Also maintains the concentration gradient as it changes the identity of the molecule and tricks glucose into flowing into the cell.

Is the first step of glycolysis reversible or irreversible. Positive or negative deltaG?

Irreversible. Negative deltaG

Second step of glycolysis

Isomerase turns glucose-6-phospate into fructose-6-phosphate→aldose to ketose

What is the purpose of turning glucose-6-phosphate into fructose-6-phosphate?

Allows the sugar to eventually be split into two equal molecules

Is the second step of glycolysis reversible or irreversible? Positive or negative deltaG?

Fully reversible. deltaG~0 or positive

Third step of glycolysis

PFK1 phosphorylates fructose-6-phosphate into fructose 1,6-biphosphate

Is the third step of glycolysis reversible or irreversible? Positive or negative deltaG?

Irreversible. Commitment step. Negative deltaG

Structure of PFK1

Multimeric enzyme that is subject to allosteric modulation

Fourth step of glycolysis

Aldolase cleaves Fructose-1,6-biphosphate into DHAP and glyceraldehyde-3-phosphate

Is the fourth step of glycolysis reversible or irreversible? Positive or negative deltaG?

Reversible. G~0 or slightly negative

Fifth step of glycolysis

Triose phosphate isomerase interconverts DHAP into glyceraldehyde-3-phosphate

Control of fifth step of glycolysis

Diffusion controlled

Is the fifth step of glycolysis reversible or irreversible?

reversible

Sixth step of glycolysis

glyceraldehyde-3-phosphate dehydrogenase turns glyceraldehyde-3-phosphate into 1,3-BPG via a redox reaction

What acts as the electron acceptor in the sixth step of glycolysis?

NAD+ gets reduced to NADH

What is the purpose of the sixth step of glycolysis?

1. Captures energy in the form of NADH that can be re-oxidized to drive glycolysis again or can be shuttled to ETC

2. Create a high energy molecule that can be used to phosphorylate ADP later down the line

What is required for the sixth step of glycolysis and where does this element come from?

Inorganic phosphate is required. This comes from the cytosol stores and DOES NOT come from ATP. No ATP is expended!

Is the sixth step of glycolysis reversible or irreversible?

reversible

Seventh step of glycolysis

Phosphoglycerate kinase catalyzes substrate level phosphorylation turning each molecule of 1,3-BPG into 3-phosphoglycerate

Is the seventh step of glycolysis reversible or irreversible?

Reversible

Eighth step of glycolysis

phosphoglycerate mutase rearranges 3-phosphoglycerate into 2-phosphoglycertae

Is the eighth step of glycolysis reversible or irreversible?

Reversible

Ninth step of glycolysis

Enolase catalyzes a dehydration reaction (forms double bond) turning 2-phosphoglycerate into PEP

Purpose of ninth step of glycolysis

The double bond in PEP activates the phosphoryl group for transfer

Is the ninth step of glycolysis reversible or irreversible?

Reversible

Tenth step of glycolysis

Pyruvate kinase catalyzes substrate level phosphorylation turning PEP into pyruvate

Is the tenth step of glycolysis reversible or irreversible?

Irreversible! Negative deltaG

Pyruvate kinase structure

Tetramer. Subject to allosteric regulation

What are the three key control steps of glycolysis?

1. Hexokinase

2. PFK1

3. Pyruvate kinase

Where does the preparatory phase end and the payoff phase begin?

1. Reaction five where triose phosphate isomerase turns DHAP into glyceraldehyde-3-P is the last step of the preparatory phase

2. Rection six where G3P gets turned into 1,3-BPG is the first payoff phase step

Feeder molecules for glycolysis

• glucose

• endogenous+dietary glycogen

• starch

• lactose

• mannose

• trehalose

two fates of pyruvate

• can be turned into Acetyl-CoA under aerobic conditions

• can be turned into lactic acid or ethanol under anaerobic condition

Forming lactate from pyruvate

Pyruvate reduced to lactate by lactate dehydrogenase with NAD+ production as a byproduct

deltaG of forming lactate

large and negative

Turning pyruvate into ethanol

1. pyruvate decarboxylated to acetaldehyde by pyruvate decarboxylase

2. acetaldehyde turned into ethanol by alcohol dehydrogenase with NAD+ as a byproduct

What is the purpose of turning pyruvate into ethanol or lactate?

Regenerate NAD+!

Gluconeogenesis

synthesis of new glucose

Relationship between gluconeogenesis and glycolysis

gluconeogenesis is the reverse of glycolysis

Does gluconeogenesis require or generate energy?

Requires energy

Three steps gluconeogenesis must work around

1. Pyruvate kinase

2. PFK1

3. Hexokinase

Steps for work around for pyruvate kinase

1. Pyruvate carboxylase attaches CO2 onto pyruvate to make OAA

2. OAA decarboxylated and phosphorylated by PEP carboxy-kinase

Requirements for pyruvate carboxylase

need ATP and biotin

Requirements of PEP carboxy-kinase

GTP

Two ways of turning OAA into PEP

• OAA is always synthesized in the mitochondria

1. turn OAA into malate, transport malate out, then turn malate back to OAA in the cytoplasm

2. turn OAA directly into PEP in the mitochondria, then transport PEP out

Work around for PFK1

fructose bisphophatase-1 turns fructose-1,6-biphosphate into fructose-6-phosphate

Workaround for hexokinase

glucose-6-phosphatase turns glucose-6-phosphate into glucose

Purpose of biotin

CO2 carrier

Regulation of hexokinase

• different isoforms (body vs liver)

• inhibited by glucose-6-phosphate

Glucokinase vs hexokinase

• glucokinase found in liver

• has 50 fold lower affinity for glucose than hexokinase

Regulation of PFK1

• inhibited by ATP and citrate

• activated by AMP, ADP, and fructose-2,6-biphosphate

Enzyme curve of PFK1

Exhibits cooperativity

• becomes sigmoidal when ATP is high→lowers affinity

• traditional curve when ATP is low

Regulation of fructose-1,6-biphosphatase

turned off by AMP→oppositely regulated

turned off by fructose-2,6-biphosphate

fructose-2,6-biphosphate

A regulator of glycolysis and gluconeogenesis→not a metabolite of either

Synthesis of fructose-2,5-biphosphate

PFK2

Breakdown of fructose-2,6-biphosphate

fructose biphosphatase-2

Regulation of fructose-2,6-biphosphate synthesis

• inhibited by glucagon

• activated by insulin

How insulin activates F-2,6-BP synthesis

• signals fed state when blood sugar is high

• induces a dephosphorylation of fructose biphosphatase-2, deactivating it

• induces activation of PFK2

How glucagon inhibits F-2,6-BP synthesis

• signals hungry state

• induces phosphorylation of fructose biphosphatase-2, activating it

• induces deactivation of PFK1

Enzyme curve of PFK1 under the influence of F-2,6-BP

F-2,6-BP greatly improves the enzyme

Enzyme curve of FBP-ase under the influence of F-2,6-BP

Inhibits enzyme

Regulation of pyruvate kinase

• inhibited by ATP, acteyl-CoA, long chain fatty acids

• activated by F-1,6-BP

Regulation of pyruvate kinase in the liver

glucagon activates PKA which phosphorylates pyruvate kinase, deactivating it

Regulation of pyruvate dehydrogenase

• inhibited by Acetyl-CoA

• stops decarboxylation of pyruvate to acteyl-coA

Regulation of pyruvate carboxylase

• turned on by Acetyl-CoA

• promotes gluconeogenesis

Pentose phosphate pathway

An offshoot of glycolysis that produces NADPH and ribose-5-phosphate

Phases of PPP

non-oxidative and oxidative

Purpose of non-oxidative phase of PPP

shuffles around carbon skeleton or molecules involved in glycolysis and/or gluconeogenesis

Purpose of oxidative phase of PPP

Produce NADPH and ribose-5-phosphate

Purpose of NADPH

used for fatty acid synthesis and precursor to glutathione which scavenges up free radicals

Purpose of ribose-5-phosphate

used in DNA and RNA synthesis

NADPH vs NADH

• NADPH drives anabolism through reductive biosynthesis

• NADH drives catabolism

Key step/enzyme of PPP

glucose-6-phosphate dehydrogenase is the commited step of PPP

Regulation of PPP

NADPH inhibits glucose-6-phosphate dehydrogenase

Explain how glycolysis and gluconeogenesis can be irreversible in the cell

Three enzymes have large, negative deltaG values. They are regulated so as to not do futile cycling

Where does the PPP occur: cytosol or mitochondria?

cytosol

Why do cells not want to export pyruvate?

Pyruvate helps regenerate NAD+

How would a defect in pyruvate kinase affect 2,3-BPG production?

Would decrease production since glycolytic intermediates build up which shuts the pathway off

What happens to the lactate that is produced during fermentation?

It is turned back into pyruvate through oxidation and eventually glucose through gluconeogenesis. That way energy is not wasted.

What is needed to turn lactate into pyruvate?

Ample NAD+

If phosphate was swapped for an unstable analog, what would occur in glycolysis?

Glyceraldehyde-3-phosphate couldn’t be turned into 1,3-bisphosphoglycerate and would instead be spontaneously hydrolyzed into 3-phosphoglycerate. This would prohibit substrate level phosphorylation down the line, meaning no net ATP could be made. This would lead to starvation and death.

Why does fermentation cease when the supply of phosphate is exhausted?

Glyceraldehyde phosphate dehydrogenase requires 2 Pi per mol glucose to produce ethanol

Why does inadequate Pi concentration lead to CO2, ethanol, and fructose-1,6-bisphosphate pooling?

Pathway backs up because Pi is used up until it is not available anymore. This causes F-1,4-BP to build up

What is niacin needed for?

Niacin is vitamin B3. It is needed to make NAD+

Why does alcohol metabolism, which uses NAD+ as a reactant cause hypoglycemia?

It creates a competition for NAD+ with gluconeogenesis. This means not enough glucose is made

Where is NAD+ used in gluconeogenesis?

In the reverse step of turning glyceraldehyde-3-phosphate into 1,3-biphosphoglycerate

How do high ADP levels increase PFK1 activity?

There is a constant amount of adenosine in the cell so high ADP means low ATP. There is an ATP binding regulatory pocket which is empty when ATP is low

Why can’t glucose-6-phosphate leave the cell?

Because it has a negatively charged phosphate on it

How is succinate glucogenic?

Can be turned into OAA

How would accumulation of a non-regulatory product in a pathway affect that pathway?

It would drive it backwards due to Le Chatelier’s principle