Glycolysis: Energy Generation Without an Oxygen Requirement Glucose Biofuel Prominence: Low-reactive...

Glycolysis: Energy Generation Without an Oxygen Requirement

Glucose Biofuel Prominence:

Low-reactive ring-form minimizes protein glycosylation

Glycolysis: A Three Step Process

• Glucose trapping and destabilization (priming)

•Three carbon unit generation (cleaving)

• Energy generation

Induced Fit in Hexokinase

Glucose induces a large enzyme conformational change

Substrate-induced cleft closing prevents ATP hydrolysis

Kinases require a divalent metal ion

What function does Mg+2 play in hexokinase?

Hexokinase Closed Around Substrates

What mechanisms of catalysis are operative?

Hexokinase Reaction Mechanism

What is the Nu:, electrophile, and leaving group in this reaction?

Phosphoglucose Isomerase: Aldose to Ketose Conversion

PGI Reaction Mechanism

Phosphoglucose Isomerase (PGI) G6P Conversion via Acid-Base Catalysis

Base catalyzed bond formation

Phosphoglucose Isomerase (PGI) G6P Conversion via Acid-Base Catalysis

Acid catalyzedketal formation

Phosphoglucose Isomerase (PGI) G6P Conversion via Acid-Base Catalysis

H+

Base catalyzes ring closure

Phosphoglucose Isomerase (PGI) G6P Conversion via Acid-Base Catalysis

Phosphofructokinase: Trapping the Fructose Isomer

What is the mechanism for this reaction?

Glycolysis Stage I: Glucose Trapping and Destabilization (priming)

Six Carbon Sugar Cleaved to Two Three Carbon Units

What is the bond to be cleaved?

Which alcohol becomes an aldehyde?

Haworth and Fischer Projections Equivalency

The functional group that is down in a Haworth projection is positioned how in a Fischer structure?

Aldolase

Fructose-1,6-bisphosphate binds to the aldolase enzyme for covalent catalysis

Aldolase Reaction Mechanism

Aldolase Reaction Mechanism

What is lost when the Schiff base forms?

Aldolase Rxn Mechanism

Aldolase Reaction Mechanism

Compare and contrast a Schiff base with a carbonyl group.

Aldolase Reaction Mechanism

What is the process for Schiff base to carbonyl conversion?

H2O

Aldolase cleaves FBP into GAP and DHAP

Aldolase Reaction Mechanism

Which previous glycolytic step is similar to TIM?

Reversible and driven towards GAP due to product depletion

Triose Phosphate Isomerase (TIM)

Glycolysis: Step #5Triose Phosphate Isomerase

DHAP conversion to GAP necessary to proceed through glycolysis

Triose Phosphate Isomerase Reaction Mechanism

TIM- or α,β-barrel with 8 parallel β-strands surrounded by 8 α-helices.

Two ATPs are initially invested.

One glucose is metabolized into two GAP molecules.

Stoichiometry: Stages 1-2 of Glycolysis

Road Map for Energy Harvest (Stage 3)

Glyceraldehyde-3-Phosphate Dehydrogenase: Covalent Catalysis

Glyceraldehyde-3-Phosphate Dehydrogenase: a 2 Step Process

What amino acid will serve as a nucleophile to form a thioester?

Glyceraldehyde-3-Phosphate Dehydrogenase: Reaction Mechanism

Glyceraldehyde-3-Phosphate Dehydrogenase: Catalysis Energetics

Actual coupled reaction

Hypothetical reaction with no coupling

What is the Nu:, electrophile and leaving group for this reaction? (hint: consider hexokinase in reverse)

Phosphoglycerate Kinase

Glycolysis: the Three Final Steps

Pyruvate Kinase

What is the Nu:, electrophile and leaving group for this reaction? (hint: consider phosphoglycerate kinase)

∆G°ʹ ∆GEnzyme (kcal/mol) (kcal/mol)

1near equilibrium means that ∆G is about zero

What is the relationship between ∆G and ∆G°ʹ?When can ∆G and ∆G°ʹ diverge?

Glycolysis Energetic

• Water represents metabolite flux

• Water amount in flask represents intermediate abundance

• Flasks connections are enzymes

• Vertical drop represents decrease in free energy

ΔG° = height difference between flask bottomsΔG = height difference between water levels

Regulating Glycolysis: A Pictorial Analogue

Metabolic Regulation

Irreversible reactions are potential regulatory sites (e.g. hexokinase, phosphofructokinase and pyruvate kinase)

What duel role does ATP play in PFK-1 catalysis?

In what direction does ATP regulate phosphofructokinase?

Energy Status Regulates Glycolytic Flow

Elevated [ATP] sufficient energy; elevate [AMP] low energy

ADP + ADP ↔ ATP + AMP <adenylate kinase>

Muscle Tissue

Fructose-2,6-Bisphosphate an Allosteric Regulator of Phosphofructokinase-1

F-2,6-BP amplifies or diminishes PFK-1 activity?

PFK-2

Liver Tissue

Front activation by fructose-6P

Fructose-2,6-Bisphosphate Reduces ATP Inhibition of Phosphofructokinase-1

ATP is a substrate and inhibitor of PFK-1

PFK-2

Liver Tissue

Fructose Entry Points for Glycolysis

Glucose + Fructose

Major dietary sugars: sucrose (table sugar) and fructose (high-fructose corn syrup)

Glycerol-3P

Fructose Metabolism

How is this different than glucose metabolism?

Lipid Synthesis

Fructose Metabolism

Glycerol 3-phosphate a precursor to triacylglycerol

Fructose catabolism bypasses phosphofructokinase regulation

Alternative Fates for Pyruvate

Anaerobic Recycling of NADH for Glycolysis

Microbial Recycling of NADH for Glycolysis

Pyruvate Dehydrogenase:

the Bridge between

Glycolysis and Citric Acid Cycle

Standard Free Energy Change Comparisons for Glucose Catabolism With and Without Oxygen

Pathogenic Obligate Anaerobes

Pyruvate Targeted for Anabolism

The biotin prosthetic group serves as a CO2 carrier

What reaction links biotin to the protein?

Pyruvate Carboxylase: an

Endergonic Reaction

Oxaloacetate

Glucose Metabolism: Both Catabolic and Anabolic

Glucose Metabolism: Both Catabolic and Anabolic

Problems: 1, 3, 5, 7, 13, and 21