Biochemistry Unit 3

kinase

kinase

an enzyme that attaches a phosphate group to a protein

isomerase

an enzyme that catalyzes the conversion of a specified compound to an isomer

mutase

moving phosphate

dehydrogenase

takes away a hydrogen

enolase

converts the molecules into an enol

aldolase

splitting of a carbon-carbon band

how much atp does glycolysis produce

two net molecules of ATP per 1 molecule of glucose

Overall glycolysis reaction

Glucose + 2 NAD + + 2 ADP + 2 Pi →2 Pyruvate + 2 NADH + 2 ATP

first step of glycolysis

hexokinase (Phosphorylation “traps” glucose in the cell)

glucose to glucose-6-p

uses ATP

second step of glycolysis

Phosphoglucoisomerase

D-glucose-6-phosphate (G-6-P) to D-fructose-6-phosphate (F-6-P)

third step of glycolysis

Phosphofructokinase

Reaction is removed from equilibrium

uses ATP

D-fructose-6-phosphate (F-6-P) to fructose-1,6-bisphosphate

Kinetics of phosphofructokinase

ATP is not only a substrate but at high concentrations is an allosteric inhibitor

fourth step of glycolysis

Fructose bisphosphate aldolase

Class I enzymes form Schiff base intermediates bound to enzyme

Class II enzymes use metal ions to stabilize the enolate intermediate

Fructose-1,6-bisphosphate to Dihydroxyacetone biphosphate (DHAP) and Glyceraldehyde-3-phosphate (G-3P)

fifth step of glycolysis

Triose phosphate isomerase

Dihydroxyacetone biphosphate (DHAP) to Glyceraldehyde-3-phosphate (G-3P)

sixth step of glycolysis

Glyceraldehyde-3-phosphate dehydrogenase

Reaction operates near equilibrium

covalent catalysis

Glyceraldehyde-3-phosphate (G-3P) to 1,3-Bisphosphoglycerate (1,3BPG)

seventh step of glycolysis

Phosphoglycerate kinase

The reaction operates near equilibrium

1,3-Bisphosphoglycerate (1,3BPG) to 3-phosphoglycerate (3PG)

eight step of glycolysis

Phosphoglycerate mutase

3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG)

ninth step of glycolysis

Enolase

2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP)

tenth of step of glycolysis

Pyruvate kinase

phosphoenolpyruvate (PEP) to pyruvate

Regulation of pyruvate kinase

• AMP, F-1,6-BP: allosteric activators

• ATP, acetyl-CoA, alanine: allosteric inhibitors

Regeneration of NAD+ (so you can do glycolysis again)

Aerobic regeneration

• Reoxidation by mitochondrial electron transport chain by mechanisms shuttling electrons into the mitochondria

Anaerobic regeneration

• Reduction of pyruvate to lactate in muscle by the enzyme lactate dehydrogenase

Decarboxylation of pyruvate to acetaldehyde (Pyruvate decarboxylase ) and reduction of acetaldehyde to ethanol in yeast by the enzyme alcohol dehydrogenase

which steps of glycolysis are regulated/steps are far from equilibrium

1, 3, 10

close to equilibrium means

delta G=0

how different sugars get metabolized so that they can enter the glycolytic scheme

• Galactose

• Mannose

• Fructose

Metabolism of glycerol

Glycerol phosphate dehydrogenase converts it to dihydroxyacetone phosphate (DHAP), a glycolytic intermediate

Citric Acid Cycle

Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi → 2 CO2 + 3 NADH + 3 H + + FADH 2 + GTP + CoA

inside mitochondria matrix

Bridging reaction

pyruvate must cross the mitochondrial membrane

Pyruvate → Acetyl-CoA

overall reaction: Pyruvate + CoA + NAD + ® Acetyl-CoA + NADH + H + + CO 2

Three prosthetic group coenzymes:

– thiamine pyrophosphate

– lipoic acid,

– FAD

“ABC’s” of the TCA Cycle

A: two carbon unit (acetyl-CoA) is Added to

four carbon carrier (oxaloacetate) to form six

carbon intermediate (citrate)

B: six carbon intermediate is Broken Down to

four carbon succinate.

C: Carrier (oxaloacetate) is regenerated from

succinate

Citrate Synthase

Reaction is removed from equilibrium (citrate is more stable than acetyl-Coa

Aconitase

near equilibrium

iron-S cluster

inhibited by Fluoroacetate

Isocitrate Dehydrogenase

removed from equilibrium

First oxidative step of the cycle.

First CO 2 produced

a-Ketoglutarate Dehydrogenase

The second oxidative step of the TCA cycle

Succinyl-CoA Synthetase

Reaction is near equilibrium

involves intermediate phosphorylation of the enzyme at a histidine residue

drive GTP synthesis

Succinate Dehydrogenase

• Third oxidative step of the cycle. Contains FAD as a covalently bound prosthetic group.

• Also contains an iron-sulfur protein.

• Reaction is near equilibrium.

Fumarase

Hydration of the double bond

Malate Dehydrogenase

Fourth oxidative step of TCA cycle

Regenerates oxaloacetate and NADH to restart the cycle

Tracing 1 Carbon Atoms

Radioactivity from 1- 14C-acetyl-CoA is not converted to CO 2 until the second turn. All of the radioactivity is lost in the second turn.

Tracing 2 Carbon Atoms

Radioactivity from 2- 14C-acetyl-CoA is not converted to CO 2 until the third turn. The rest remains in the cycle intermediates, losing one-half on each subsequent turn.

Malate shuffle

smuggle the electrons on malate, malate is able to go to the mitochondrial matrix . citrate can go out to the cytosol

Anaplerotic Reactions

1. Pyruvate carboxylase

2. PEP carboxylase (primarily in plants)

3. Malic enzyme

4. PEP carboxykinase

Pyruvate Carboxylase co factor

Contains biotin prosthetic group

Of the eight TCA reactions, only three are far removed from equilibrium

– Citrate synthase

– Isocitrate dehydrogenase

– a-Ketoglutarate dehydrogenase

note: bridging reaction also is removed form equilibrium

Regulation of Pyruvate Dehydrogenase

• inhibited by acetyl-CoA

• inhibited by NADH

• inhibited by GTP

• activated by NAD+ and CoA

Regulation of Citrate Synthase

• Inhibited by ATP

• Inhibited by NADH

• Inhibited by succinyl CoA

Regulation of Isocitrate Dehydrogenase

• Inactivated by NADH

• Inactivated by ATP

• activiated by ADP

Regulation of a-Ketoglutarate Dehydrogenase

• Succinyl-CoA inhibits

• NADH inhibits

• AMP activates

Glyoxalate Cycle

Two additional enzymes

Isocitrate lyase

Malate synthase

(only in plants)

Oxidative Phosphorylation

The proton gradient runs downhill to drive the synthesis of ATP

Gibbs free energy

• Delta G is the standard change in Gibb's free energy

• n is the number of electrons involved in the redox reaction

• F is Faraday's constant (can be found on equation sheet),

• E is the standard cell potential (measured in volts)

What is the terminal electron acceptor in electron transport?

O2

Membrane bound complexes

Complex I: NADH-Coenzyme Q Reductase;

Complex II: Succinate-UQ reductase;

Complex III: UQ-Cyt c reductase;

Complex IV Cytochrome c oxidase

Water soluble complexes

Cytochrome c

Flavoproteins

Contain tightly bound FMN or FAD, one or two electron transfer

Coenzyme Q

Also called ubiquinone (CoQ or UQ). One or two electron transfer

Cytochromes

Contain heme. One electron transfer

Iron-sulfur proteins

Transfer electron involving Fe 2+

Protein-bound copper

Transfer electron involving Cu+

Electron transport complexes far removed from equilibrium

Complex 1,3,4

Complex I cofactors

• Ubiquinone (UQ; also known as coenzyme Q; CoQ)

• Iron-sulfur

• FMN

• NADH

Complex II cofactors

• UQ

• FAD

Complex III cofactors

• UQ

• FeS

Q cycle

UQH2 +2 H+in +2 Cyt c ox +2e- → 4 H+ + 2cyt red + UQ

Complex IV cofactors

• CU

• iron

  4cyt c red + 4H + +O 2 4cyt c ox + 2H 2 O

A model for the overall electron transport path

The mitochondrial matrix is negative (high pH) and the intermembrane space is positive (low pH) due to the electron transport chain

what subunit rotates? What subunit changes confirmations?

gamma rotates,

beta changes conformations

ATP/ADP Translocase

Mediates Transport Across the MM to cytosol

Glycerol Phosphate Shuttle

Couples The Redox Reactions

malate-aspartate system

instead of moving entire NADH, we will just move the H- (hydride) to and from the cytosol and mitochondrial matrix