Pratt and Cornely, Chapter 13 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 ATP ATP Pi + NAD+ 2x ADP ADP NADH ADP ADP 2x 2x 2x ATP ATP Hexose and triose phases Energy input and payoff phases

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 Amino acid and nitrogen metabolism Gluconeogenesis Aerobic Energy Anaerobic in higher organisms Anaerobic in microorganisms

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

Glucose Metabolism Overview Gluconeogenesis Glycogen metabolism Pentose Phosphate Pathway

Precursors for Gluconeogenesis Names of compounds? Type of reaction? Type of enzyme? Cofactor(s)? More on lactate processing later…

Chemistry of Gluconeogenesis Chemically opposite of glycolysis (mainly) Energetically costly—no perpetual motion machine! Points of regulation

Glycolysis Gluconeogenesis Step 1: costs 1 ATP Step 10: no change 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 Hormone 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

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

Chemistry of Degradation Glycogen phosphorylase Key Regulation site Inorganic phosphate as a nucleophile Remake G-1-P with no ATP cost

Overall Energetics

Key Enzymes

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

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.

Summary of glucose metabolism