Glucose Metabolism

Pratt and Cornely, Chapter 13

Glycolysis Expectations

• Memorize/learn Figure 13.2• Know overall reaction and stages• Explain chemical logic of each step• Enzyme mechanisms presented in book

Glycolysis

• Ten enzymes that take glucose to pyruvate

• Cytosol• ATP and NADH

Reactions and Enzymes of Glycolysis

• Hexose and triose phases

• Energy input and payoff phases

ATP ATP

ADP ADP2x

Pi + NAD+

NADH

2x

ADP ADP

ATP ATP

2x 2x

Energy Input

Energy Payoff

Know...

• Substrates • Co-substrates • Products • Enzyme names

1. Hexokinase

• Previous concepts: Induced fit, kinase• Energy use/production? • Chemical logic?

Problem 3• (Notice miswording) The DGo’ value for

hexokinase is -16.7 kJ/mol, and the DG value under cellular conditions is similar.– What is the ratio of G-6-P to glucose under standard

conditions at equilibrium if the ratio of ATP:ADP is 10:1?

– How high would the ratio of G-6-P to glucose have to be to reverse the hexokinase reaction by mass action?

2. Phosphoglucose Isomerase• Previous concepts: Isomerization• Energy use/production? CONCEPT: Near-equilibrium• Chemical logic?• Stereochemistry—reverse does not produce mannose!

3. PFK-1• Previous concepts: Allosteric inhibition• Energy use/production? • Chemical logic?• First committed step of glycolysis

– Why?– regulation

Regulation

4. Aldolase• Previous concepts: Standard free energy is +23kJ, but it

is a near equilibrium reaction• Energy use/production? • Chemical logic?• Beginning of triose stage

Aldolase Mechanism

5. Triose Phosphate Isomerase• Previous concepts: Catalytic perfection• Energy use/production? • Chemical logic?• Most similar to which previous reaction?

6. Glyceraldehyde-3-P DH• Previous concepts: Redox and

dehydrogenase• Energy use/production? • Chemical logic?

GAPDH Mechanism

7. Phosphoglycerate Kinase • Previous concepts: High energy bond• Energy use/production?

– Substrate level phosphorylation• Chemical logic?• Coupled to reaction 6

Coupled Reactions

• GAPDH = 6.7 kJ/mol• PG Kinase = -18.8 kJ/mol• Overall:

8. Phosphoglycerate Mutase• Previous concepts: Covalent catalysis• Energy use/production? • Chemical logic?• Mutase—isomerization with P transfer

Mechanism

• Not a simple transfer• What happens if the bisphosphate escapes?

9. Enolase• Concept: Phosphoryl group transfer potential• Energy use/production? • Chemical logic?

10. Pyruvate Kinase• Energy use/production? • Chemical logic?• Regulation: F-1,6-BP can act as a feed-

forward activator to ensure fast glycolysis

Overall Energetics

• Standard Free energies are up and down

• Free energies under cellular conditions are downhill – Three irreversible

Fate of Pyruvate

Aerobic Energy

Anaerobic inmicroorganisms

Anaerobic inhigher organisms

Gluconeogenesis

Amino acidand nitrogenmetabolism

The Problem of Anaerobic Metabolism

• With oxygen, the NADH produced in glycolysis is re-oxidized back to NAD+

• NAD+/NADH is a co-substrate which means…• If there is no oxygen, glycolysis will stop

because…• The solution to the problem is to…

The solution in Yeast• Pyruvate is decarboxylated

(cofactor?) to acetaldehyde• Acetaldehyde transformed to

ethanol – What type of reaction?– What cofactor?

• NAD+ is regenerated to be reused in GAPDH

The Solution in Us

• Lactate formation

• Balanced equation

We don’t operate anaerobically...

• Most energy still trapped in lactate

• Back to pyruvate, then acetyl-CoA

• Citric acid cycle

Other sugars enter glycolysis

High fructose diet puts sugars through glycolysis while avoiding major regulation step

Glucose Metabolism Overview

• Keep the main pathway purposes distinct

• But learn details of chemistry and regulation based on similarities

O

HO

HO

OH (P)

OH

OH

DHAP

Pyruvate

Gluconeogenesis

Lactate

Amino Acids

Glycerol(Triacylglycerides)

Glycogen

Glycogen Degradation

Glycogen

Glycogen Synthesis

Ribose,NADPH

ATP

DHAP

Pyruvate

Pentose Phosphate Pathway

Energy Production

Starch

Diet

Glucose Metabolism Overview

• Gluconeogenesis• Glycogen

metabolism• Pentose

Phosphate Pathway

O

HO

HO

OH (P)

OH

OH

DHAP

Pyruvate

Gluconeogenesis

Lactate

Amino Acids

Glycerol(Triacylglycerides)

Glycogen

Glycogen Degradation

Glycogen

Glycogen Synthesis

Ribose,NADPH

ATP

DHAP

Pyruvate

Pentose Phosphate Pathway

Energy Production

Precursors for Gluconeogenesis

• Names of compounds?

• Type of reaction?• Type of enzyme?• Cofactor(s)?• More on lactate

processing later…

OH

OH

OH

OPO3

O

OH

O

O

O

NH2

O

O

O

O

O

OH

O

O

Chemistry of Gluconeogenesis

• Chemically opposite of glycolysis (mainly)• Energetically costly—no perpetual motion

machine!• Points of regulation

Glycolysis• Step 1: costs 1 ATP• Step 3: costs 1 ATP• Step 7: makes 2 ATP• Step 10: makes 2

ATP

• Gluconeogenesis• Step 10: no change• Step 8: no change• Step 3: costs 2 ATP• Step 1: costs 4 ATP

equivalents

Step 1a

• Pyruvate Carboxylase– Biotin– Costs ATP to make driving force for next reaction– First step in biosynthesis of glucose and many

other molecules• Related to which amino acid?

Mechanism

• Mixed anhydride• Coupled through

biotin coenzyme

Step 1b

• PEP carboxykinase– ATP cost to restore PEP– CO2 loss drives rxn

Step 8• Fructose-1,6-bisphosphatase• No additional energy input• Phosphate ester hydrolysis is spontaneous

Step 10

• Glucose 6-phosphatase– Liver (and others)– Not in muscle

Problem 34

• A liver biopsy of a four-year old boy indicated that the F-1,6-Bpase enzyme activity was 20% normal. The patient’s blood glucose levels were normal at the beginning of a fast, but then decreased suddenly. Pyruvate and alanine concentrations were also elevated, as was the glyceraldehyde/DHAP ratio. Explain the reason for these symptoms.

Key Regulation• At the committed step in glucogenic cells• Principle of Reciprocal regulation• Local regulation vs Hormone regulation

Key Regulation

• Local regulation– AMP/ATP (energy charge)– Citrate (feedback)

• Hormone regulation– Fructose-2,6-bisphosphate

• Gluconeogenesis is inhibited• Glycolysis is stimulated

Problem 39

• Brazilin, a compound found in aqueous extracts of sappan wood, has been used to treat diabetics in Korea. It increases the activity of the enzyme that products F-2,6-BP and stimulates the activity of pyruvate kinase. What is the effect of adding brazilin to liver cells in culture? Why would brazilin be an effective treatment for diabetes?

Glucose Metabolism Overview

• Gluconeogenesis• Glycogen

metabolism• Pentose

Phosphate Pathway

O

HO

HO

OH (P)

OH

OH

DHAP

Pyruvate

Gluconeogenesis

Lactate

Amino Acids

Glycerol(Triacylglycerides)

Glycogen

Glycogen Degradation

Glycogen

Glycogen Synthesis

Ribose,NADPH

ATP

DHAP

Pyruvate

Pentose Phosphate Pathway

Energy Production

Glycogen

• Storage molecule• Primer necessary• Very large!• Multiple ends allow

for quick synthesis and degradation

Chemistry of Synthesis

• Step 1

• Near equilibrium• The link to glucose-6-phophate, our central

molecule

Chemistry of Synthesis• Step 2• Count high energy

bonds• Pyrophosphatase

– Common motiff• UDP-glucose:

activated for incorporation

Chemistry of Synthesis• Step 3• Glycogen

synthase• Growing end is

non-reducing• UDP released

Energetics of Synthesis

• Total cost: one ATP equivalent from G-6-p

O

HO

HO

O

OH

OH

P

O

O O

O

HO

HO

O

OH

OH

P-P-Uridine

O

HO

OH

OH

OH

O

O

OH

HO

O

HO

Glucose-6-P

UDP

UTP

2 Pi

Chemistry of Degradation

• Glycogen phosphorylase

• Key Regulation site• Inorganic phosphate

as a nucleophile• Remake G-1-P with

no ATP cost

Overall Energetics

O

HO

HO

O

OH

OH

P

O

O O

O

HO

HO

O

OH

OH

P-P-Uridine

O

HO

OH

OH

OH

O

O

OH

HO

O

HO

Glucose-6-P

UDP

UTP

2 Pi

Pi

Key Enzymes

O

HO

HO

O

OH

OH

P

O

O O

O

HO

HO

O

OH

OH

P-P-Uridine

O

HO

OH

OH

OH

O

O

OH

HO

O

HO

Glucose-6-P

UDP

UTP

2 Pi

Pi

Glycogen Synthase

Glycogen Phosphorylase

Glycogen Storage Diseases

Many disrupt glycogen breakdown in muscle and/or liver (hypoglycemia, enlarged liver, muscle cramps...)

Glucose Metabolism Overview

• Gluconeogenesis• Glycogen

metabolism• Pentose

Phosphate Pathway

O

HO

HO

OH (P)

OH

OH

DHAP

Pyruvate

Gluconeogenesis

Lactate

Amino Acids

Glycerol(Triacylglycerides)

Glycogen

Glycogen Degradation

Glycogen

Glycogen Synthesis

Ribose,NADPH

ATP

DHAP

Pyruvate

Pentose Phosphate Pathway

Energy Production

Pentose Phosphate Pathway

• Dual Purpose– Synthesis of “reducing potential”– Synthesis of 5-carbon sugars

• At cost of one carbon worth of carbohydrate• Net reaction:

Complex, 2-Stage Process

• Oxidative Stage– Generates reducing

power and ribose• Non-oxidative stage

– Regenerates 3- and 6-carbon sugars from 5 carbon sugars

Oxidative Stage Step 1:

• G-6-P DH• Lactone formation

Oxidative Stage Step 2:

• Also a spontaneous hydrolysis• Practice mechanism, carbohydrate orientation

Oxidative Stage Step 3:

• Oxidative decarboxylation• We will see this process again

Biosynthesis of Ribose

Non-oxidative Stage• To understand purpose, realize that we

generally need to make much more NADPH than ribose

• Problem: stuck with C5, but need C6 and C3• Solution: “Shunt” C5 back to C6 through near-

equilibrium reactions

PPP Reactions

• Epimerase• Isomerase• Transketolase• Transaldolase

Transketolase• Use cofactor (B1) to overcome chemical problem

Mechanism

Different Modes for Different Purposes

Problem 58

• A given metabolite may follow more than one metabolic pathway. List all possible fates of glucose-6-P in (a) a liver cell and (b) a muscle cell.

Summaryof glucosemetabolism