Biochem 501: Unit 3 - Bioenergetics and Catabolism

Su2026 Biochem 501 - Prof. R. Amasino - UW-Madison

Oxidation of ____ and ____ bonds result in energy production by a variety of things

C—H and C—C

Basis of large free energy change - favorable (-∆Gº)

ATP hydrolysis

ATP usually provides energy by…

Group transfers, not by direct hydrolysis

Step 1 of Glycolysis (preparatory stage)

Hexokinase reaction — glucose phos “coupled” to ATP hydrolysis (-∆G)

Step 2 of Glycolysis (preparatory stage)

Phosphohexose Isomerase — glucose to fructose isomerization, enables cleavage between C3 and C3 resulting in two 3C sugards at aldolase step

Step 3 of Glycolysis (preparatory stage)

Phosphofructokinase (PFK-1) - ATP is substrate, but inhibits enzyme activity because binds to both active site and separate allosteric site (-∆G)

Step 4 of Glycolysis (preparatory stage)

Aldolase - hexose —? 2 trioses (+∆G, but negative when actual pathway is operating)

Step 5 of Glycolysis (preparatory stage)

Triose Phosphate Isomerase (TPI) - isomerization of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate (+∆G)

Step 6 of Glycolysis (preparatory stage)

Oxidation of Glyceraldehyde-3-P - dehydrogenase reaction (only redox reaction in glycolysis, NAD+ → NADH (reduced)

Step 7 of Glycolysis (payoff stage)

Phosphoglycerate Kinase - couples to substrate level phosphorylation (-∆G)

Step 8 of Glycolysis (payoff stage)

Phosphoglycerate Mutase - rearrange phosphate so is in a high energy stage (+∆G)

Step 9 of Glycolysis (payoff stage)

Formation of phosphoenolpyruvate — similar to step 6 (+∆G)

Step 10 of Glycolysis (payoff stage, second payoff)

ATP from PEP: pyruvate kinase (-∆G)

Energy changes in glycolysis — Glucose → 2 pyruvate coupled to…

2 ADP + 2Pi → 2 ATP

2 NAD+ → 2 NADH

Irreversible steps of glycolysis

Step 1 (hexokinase), Step 3 (PFK-1), and Step 10 (pyruvate kinase)

Fermentations

Ways to anaerobically regenerate NAD+ from NADH to maintain glycolysis

Pyruvate to lactate

lactate dehydrogenase reaction, less energy provided when fully oxidized

pyruvate to ethanol

most energy provided when fully oxidized

Pyruvate fate

Produced in glycolysis to be oxidized to acetyl-CoA to be used in citric acid cycle

Pyruvate dehydrogenase complex

enzyme complex with 3 subunits, inhibited by NADh, ATP, and Acetyl Co-A, stimulated by NAD+, AMP, and CoA

Lipoic acid

Acyl- and redox carrier — on subunit 2, facilitates shuttling substrate

Thiamine Pyrophosphate (TPP)

Can act as an anion that is involved in the reaction — could activate peptide bond to intiate cleavage

PDH - E1 rxn

Decarboxylation - covalent intermediate resolves, releases CO2

PDH - E2 rxn

Oxidation - releases acetyl Co-A

PDH - E2 rxn

Shuttling electrons to carrier (NAD+) and enabling PDH to go another round

Citric Acid Cycle (at a glance)

Final phase of aerobic path of Glucose → Pyruvate → Acetate → CO2

Central energy yielding path — point of convergence of catabolism of fats, carbs, protein

Reduced coenzymes — reducing power

Source of precursors for biosynthesis

Input and Output of Citric Acid Cycle

Input: Acetate of Acetyl-CoA, (2 C and 4 reduced bonds)

OutputsL 3 NADH, 1 FADH2, 2 CO2, and 1 GTP

Step 1 of Citric Acid Cycle

Citrate synthase - regulated step, driven by -∆G from hydrolysis of CoA, which is recycled

Step 2 of Citric Acid Cycle

Aconitase - isomerization (+∆G)

Step 3 of Citric Acid Cycle

Isocitrate Dehydrogenase - catalyzes progressive oxidations, decarboxylation of 6C → 5C

Step 4 of Citric Acid Cycle

Alpha-ketoglutarate Dehydrogenase Complex - oxidative decarboxylation, some energy preserved in thiolester (like in PDH), same cofactors (thiamine, lipoate), similar E1 and E2 rxns, identical E3

Step 5 of Citric Acid Cycle

Succinyl CoA synthetase - harvest energy of thiolester via substrate-level phosphorylation → GTP formed which is convertible to ATP

Step 6 of Citric Acid Cycle

Succinate Dehydrogenase - desaturation oxidation, e- acceptor FAD covalently bound to enzyme gets reduced to FADH2

Step 7 of Citric Acid Cycle

Fumarase - hydration reaction, prepares substrate for final oxidation

Step 8 of Citric Acid Cycle

Malate Dehydrogenase - higher endergonic, product removal by exergonic citrate synthase rxn ensures that concentration of oxaloacetate is low when cycle runnning

Regulation of Citric Acid Cycle

Inhibited by ATP, acetyl-CoA, NADH, fatty acids,

Lipids

general term which includes membrane components, hormones, vitamins, etc.

Triacylglycerols

Type of lipid used for energy storage

Bile salts

detergents — emulsification

Lipases

hydrolysis to fatty acids which are the form transported across the plasma membrane

Fatty Acid Oxidation Steps

1. Activation: fatty acid joined to CoA - enzymes on outer mitochondrial membrane

2. Transport across inner mitochondrial membrane into mitochondrial matrix- carnitine carrier system allows this

3. Beta-oxidation - conversion of fatty acid into acetyl-CoA units in mitochondrial matrix

Fatty Acid Oxidation - Activation

Fatty acids are activated by acyl-CoA synthetases,

Beta-oxidation Steps

1. Dehydrogenation

2. Hydration

3. Dehydrogenation (again)

4. Thiolate cleavage

Cofactors

NAD, FAD, and CoA have one end to carry electrons or chemical units and the other to base-pair at the active site of a ribozyme, which are RNA enzymes with catalytic bdining pockets

Using amino acids as fuel

Generates toxic ammonia, which is eliminated in mammal’s urine as urea (produced in the liver)

Aminotransferases

Catalyze the removal of an amino group which forms keto acids via transainations (swapping amino groups)

Rxns always involve glutamate and alpha-ketoglutarate

Transamination

Generates keto acids for citric acid cycle in the lvier and glutamate which can be used for urea formation

Liver as the site for urea production

Nitrogen flows to urea cycle via amino acids to glutamine and glutamate, then to NH4+ and asprate

• N flows from glutamine and glutamate to NH4+ via glutaminase and glutamate dehydrogenase

• N flows from glutamate to aspartate via aspartate aminotransferase

Regulation of glutamate dehydrogenase

ADp and GTP modulate, so when GTP is low and ADP is high, needs alpha keto acids for energy via citric acid cycle and/or gluconeogenesis

Pathway for adding NH4+ to CO2

Synthesis of Carbamoyl Phosphate - ATP used to create high-energy intermediates to drive reaction—starts the Urea cycle basically

Urea Cycle

Brings the Nitrogens together

In cytoplasm, citrulline to argininosuccinate, then arginine to ornithine which can participate in another round of the cycle

Mitochondrial electron transport and ATP production

In Eukaryotes, e- from NADH and FADH2 to O@ occurs in mitochondria via electron transport chain

Electron-transfer circuits from NADH to O2 and succinate/FADH2 to O2

NADH through I → Q → III → Cyt C → IV → O2

Succinate/FADH2 through II → Q → III → Cyt C → IV → O2

How NADH and FADH2 are used to make ATP

Electro motive force (reducing power) is converted to a proton-motive force (proton gradient) and then into higher-energy phosphate bonds (ATP)

Ubiquinone (Co-enzyme Q)

A membrane soluble mobile carrier

Complex I: NADH Ubiquinone Oxidoreductase

Catalyzes oxidation of NADH and reduction of UQ and pumps 4 H+

Complex II: Succinate Dehydrogenase

Catalyzes oxidation of succinate (with FADH2 as bound cofactor) and reduction of UQ - only membrane-inserted enzyme of citric acid cycle, no H+ pumping, only increases pool of ubiquinol

Complex III: Ubiquinol-cytochorme c odixoreductase

Oxidizes ubiquinol and reduces Ct. c and pumps 4H+ per 2e- transferred to 2 cyt c

Cytochrome C (Cyt C)

A protein that shuttles electrons between complexes III (reduced by) and IV (oxidized by)

Complex IV: Cytochrome oxidase

Complex at which O@ is consumed by respiring organisms

Accumulates 4 e- from 4 cyt c and reduces O2 to 2H2O

ATP synthesis

Chemiosmotic — electron flow coupled to formation of a gradient of H+ across the mitochondrial inner membrane

Coupling in ATP synthase

Protons (H+) will not flow through ATP synthase unless the substrates (ADP & Pi) are present

Rotational Catalysis: How H+ flux drive ATP synthesis

Bound ATP from from ADP + Pi without energy input from the proton gradient, conformation change to release ATP requires energy of the proton gradient via ɣ subunit rotation

Light reactions

light energy + H2O → chemical energy (NADHPH + ATP) + O2

Calvin cycle: carbon fixation reactions

chemical energy (NADPH + ATP) + CO2 → sugars

NADPH and ATP used to reduce C—O bonds

Methanogens

Part of the reason for why earth didn’t free when 3 bya the sun was at 70% of the “shine” it has now

Photosynthesis

Source of O2 for atmopshere

Uncouplers (ATP synthesis)

membrane permeable H+ carriers which dissipate proton gradient which reduce o rprevent ATP synthesis, but actually can speed up e- transport (causing you to lose weight)