BCMB 401 Exam 3 Learning Objectives/Review

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How is phosphorylation used to regulate protein activity

adds a phosphate group to amino acid residues, acting as a reversible on/off switch

Describe the mechanism used to activate protein Kinase A

1. signal (adrenaline, glucagon) binds to GPCR activates G-protein stimulating the enzyme adenylyl cyclase converting ATP into cAMP increasing concentration inside cell

2. PKA is inactive tetramer (two regulatory and two catalytic subunits) and active sites are blocked remaining off

3. Four cAMP molecules bind to regulatory subunits causing allosteric conformational change

4. Conformational change lowers the affinity between the regulatory and catalytic subunits, the complex dissociates releasing two catalytic subunits

5. Active catalytic subunits phosphorylate Ser, Thr residues on target proteins altering activity

Define signal transduction

Release of diffusible secondary messengers and activation of protein kinases

Describe the common elements of signal transduction pathways (The Signal Transduction Paradigm)

• Signal: hormone or environmental signal that binds to a complementary target receptor

• Receptor: membrane protein recognizing the signal, ligand bonding alters receptor shape

• Transduction: release of diffusible secondary messengers and activation of protein kinases

  • Amplification: single component activates multiple downstream molecules, multiplying strength/output

• Response: cellular changes in metabolism leading to response to original signal, regulatory mechanisms shut pathway down when signal is removed

Identify common second messengers

cAMP (targets PKA), DAG and PIP3 (targets PKC and Akt), Ca²⁺ (regulatory proteins, activate downstream protein kinases)

Describe B-AR signaling in the context of the Signal Transduction Paradigm

• Reception: epinephrine binds to ß-ARs (7TM receptor), stimulates binding of ß-Arrestin blocking activation of additional G-proteins

• Dissociation of epinephrine, phosphorylation on receptor kinase with recruits ß-Arrestin, blocking site from G-proteins

Describe the steps of GPCR signaling through protein kinase A

• Signaling molecule to GPCR, conformational change interacting with heterotrimeric G-protein causing α subunit to release bound GDP and bind fresh GTP molecule.

• Binding GTP activates α subunit, dissociates from ßgamma complex and receptor

• Active α-GTP subunit diffuses along membrane and activates enzyme adenylyl cyclase which catalyzes conversion of ATP into cAMP

• cAMP binds to regulatory subunits of PKA, detaching and releasing the active catalytic subunits of PKA - phosphorylating proteins/enzymes

Describe the steps of GPCR signaling through protein kinase C

• extracellular ligand bvinds to GPCR, structural change to receptor. Heterotrimeric G protein α subunit releases GDP and binds GTP causing G-protein to split and moves along inner membrane

• α subunit binds to and activates PLC enzyme cleaving PIP2 into IP3 and DAG

• IP3 to ER releasing stored Ca²⁺ to cytoplasm

• DAG recruits PKC and spike in calcium helps fully activate PKC

• PKC phosphorylates target proteins and transcription factors

Explain how GPCR signaling is terminated

• α subunit has intrinsic GTPase activity that hydrolyzes GTP back to GDP, reassociating with dimer and turn off downstream effectors

• ß-arrestin recruitment to phosphorylated GPCR blocks receptors

• Phosphatases remove phosphate groups with downstream PKC or PKA returning target proteins to inactive states

Identify second messengers and amplification steps in GPCR signaling

• One GPCR can catalyze activation of multiple heterotrimeric G-protein

• Each α subunit binds to and activates adenylyl cyclase/PLC which produces cAMP/IP3

• cAMP or Ca²⁺ activate protein kinases, each phosphorylates and activate many downstream proteins/enzymes

Describe Insulin receptor signaling in the context of the Signal Transduction Paradigm

• Insulin on α-subunit of insulin receptor, conformational shift causing cross phosphorylation of Tyr within activation loop “on” on ß-subunits

• ß-subunits high affinity for IRS protein, phosphorylating IRS on multiple tyrosine residues, binding PI3K to membrane converting PIP2 into PIP3

• PIP3 recruits PDK1 and Akt

• Increase glucose transport on cell surface

Explain how insulin receptor signaling is terminated

• Protein Tyrosine Phosphatases (PTPs) remove phosphate group from IRS resetting to inactive state

• Lipid phosphatases dephosphorylate PIP3 back to PIP2

Identify commonly used activated carriers

ATP, NADH, FADH2, GTP, Acetyl-CoA, Biotin

Describe the use of activated carriers, what is meant by activated

store and transfer energy to couple energetically unfavorable reactions with favorable breakdown processes

Define gluconeogenesis

occurring in liver, gluconeogenesis synthesizes new glucose molecules to maintain blood glucose levels

Identify the enzymes used in gluconeogenesis

Pyruvate carboxylase, PEP carboxykinase, fructose 1,6-biphosphatase, and glucose-6-phostphatase

Identify the intermediates of glulconeogenesis

Pyruvate

Phosphoenolpyruvate

2-phosphoglycerate

1,3-Biphosphoglycerate

Glyceraldehyde 3-phosphate

Dihydroxyacetone phosphate

Fructose 1,6-biphosphate

Fructose 6-phosphate

Glucose 6-phosphate

Glucose

Explain the importance of biotin to gluconeogenesis

biotin is a required cofactor to pyruvate carboxylase

Explain how energy is supplied for gluconeogenesis

high concentrations of ATP and acetyl-CoA inhibit pyruvate kinase and activate gluconeogenesis

Compare/contrast regulation of glycolysis and gluconeogenesis in muscle and in the liver

• High glucose -

• Low glucose -

• High AMP, High ADP, low ATP -

• High ATP -

• High Acetyl-CoA/Citrate

• High glucose - glycolysis

• Low glucose - gluconeogenesis

• High AMP, High ADP, low ATP - Glycolysis

• High ATP - Gluconeogenesis

• High Acetyl-CoA/Citrate - Gluconeogenesis

Explain why it would be unfavorable for glycolysis and gluconeogenesis to be active at the same time. Define reciprocal regulation and explain its importance

If they were running at the same time it would waste energy as a futile cycle

Contrast the positive and negative regulators of glycolysis and gluconeogenesis

• Glucagon

• Insulin

• F 2,6 BP

• Glucagon - gluconeogensis

• Insulin - glycolysis

• F 2,6 BP - glycolysis

Explain how the activity of PFK-2/FBPase-2 is regulated

• Glucagon activates PKA phosphorylates Ser 32 and inactivates PFK-2 domain, activating FBPase-2 turning on gluconeogenesis

• Insulin dephosphorylates PKA, activating PFK-2 domain and inavtivates FBPAse-2 domain, stimulating glycolysis

Define fermentation

low energy yield, cytosol, 2 ATP per glucose, anaerobic

Describe the steps of lactic acid and ethanolic fermentation

• Lactic Acid - NADH accumulates and NADH re-oxidized to sustain glycolysis with lactate dehydrogenase

• Ethanolic - NADH recycled by dual action of pyruvate decarboxylase and alcohol dehydrogenase

Explain how pyruvate is activated to produce acetyl-CoA

Through PDH

• TPP initiates decarboxylation in E1

• Carboxyl group is removed from pyruvate releasing CO2

• Hydroxyethyl group is oxidized to ecetyl group, used to reduce lipoamide group on E2

• Acetyl group transferred from lipoamide to CoA-SH creating acetyl-CoA

• E3 reoxidizes lipoamide by passing electrons to FAD (riboflavin) then to NAD+ (niacin)

Write the overall reaction for conversion of pyruvate to acetyl-CoA

Pyruvate + CoA-SH + NAD⁺ → NADH + Acetyl-CoA + CO2

Identify the cofactors required by the PDH complex

TPP, Lipoamide, Coenzyme A, FAD, NAD

Explain the energetics of the PDH reaction, what drives the formation of the thioester bond of Acetyl-CoA and what forms of “useful energy” are produced

energy is stored in reducing power as NADH and thioester Acetyl CoA.

Breaking of disulfide bonds drives energetics

Describe the general steps of the Citric Acid Cycle in terms of order of steps, reaction types, number, and type of energy-carrying molecule formed or used, number of carbons of intermediates, and cofactors used

1. Oxaloactate + Acetyl-CoA → Citrate

   1. Condensation by Citrate synthase

   2. Release CoA

2. Citrate → Isocitrate

   1. Isomerization by Aconitase

3. Isocitrate → alpha-ketoglutarate

   1. Oxidative decarboxylation by isocitrate dehydrogenase

   2. Release CO2 and NADH

   3. ATP and NADH inhibitors

4. alpha-ketoglutarate → succinyl-CoA

   1. Oxdiative decarboxylation by alpha-ketoglutarate dehydrogenase

   2. Release CO2 and NADH

   3. ATP, NADH, and succinyl CoA inhibitors

5. Succinyl-CoA → Succinate

   1. Thioester cleavage and Phosphorylation by Succinyl CoA synthetase

   2. Release ATP

6. Succinate → Fumarate

   1. Oxidation by Succinate dehydrogenase

   2. Release FADH₂

7. Fumarate → Malate

   1. Hydration by Fumerase

8. Malate → oxaloacetate

   1. Oxidation by Malate Dehydrogenase

   2. Release NADH

Describe the regulation of the PDH complex

• Activated by high NADH, Acetyl CoA, ATP concentrations, Ca²⁺

• Inhibited by pyruvate and ADP

Describe the feedback loops that regulate citric acid cycle enzyme activity

Order the steps of the cell signaling paradigm

1. Signal (first messenger)

2. Reception

3. Transduction & Amplification(second messengers)

4. Response

Which of the following is not a component of a typical signaling cascade: receptor activation, secondary messengers, protein phosphorylation, orATP synthesis

ATP synthesis

What enzyme reverses phosphorylation by removing a phosphate group from a protein?

Protein phosphatase

Inactive protein kinase A (PKA) contains

two catalytic subunits and two regulatory subunits

What part of the signaling pathway do 7TM receptors contribute to

Transduction

What is the role of G-proteins in signal transduction

Transmit signals from the receptor to other intracellular molecules

In the epinephrine signaling pathway, which of the following is activated by the beta-adrenergic receptor

Adenylate cyclase

7TM/GPCR Signaling Cascade Example: Epinephrine Receptor with Adenylate Cyclase

Beta-AR is a 7TM receptor coupled to G-proteins. Ligand binding leads to G-protein activation wherein GDP switched for GTP. Dissociation of alpha subunit from the beta-gamma subunits. Galpha activates adenylate cyclase which converts ATP to cAMP

Alternative downstream signaling cascade: Epinepherine Receptor with Phospholipase C

Activated G protein activates PLC, which cleaves PIP2 to generate DAG and IP3. DAG activates PKC, and IP3 stimulates calcium release from ER. Signaling through PLC can occur downstream of GPCRs or RTKs

Receptor Tyrosine Kinase (RTK) Signaling Cascade Example: Insulin Receptor

IRS bridges insulin-activated receptor to PI3K, which synthesizes PIP3, which activates PDK1, which activates AKT. AKT stimulates glucose uptake by the cell

What is the primary role of catabolic pathways

to extract energy from fuels

Where does glycolysis occur in the cell?

Cytoplasm

Glycolysis Stage 1 The Investment Stage Summary

During stage 1 of glycolysis, two molecules of ATP are invested to convert glucose into fructose 1,6-biphosphate in three steps: a phosphorylation, an isomerization, and a second phosphorylation reaction

Order of reactions from stage 1 of glycolysis

1. Hexokinase

2. Phosphoglucose isomerase

3. Phosphofructokinase

4. Aldolase

5. Triose phosphate isomerase

What enzyme catalyzes the first committed step of glycolysis

Phosphofructokinase

What net ATP per molecule of glucose is associated with stage 1 of glycolysis

use 2 ATP

What net ATP per molecule of glucose is associated with stage 2 of glycolysis

generate 4 ATP

Glycolysis Stage 2 The Payoff Stage Summary

Occurs twice for each molecule of glucose transformed in stage 1. Glyceraldehyde-3-P is converted to pyruvate and 2 ATP molecules are generated along with an NADH. Glycolysis nets 2 ATP and 2 NADH for every glucose unit

Order of reactions from stage 2 of glycolysis

1. Glyceraldehyde-3-phosphate dehydrogenase

2. Phosphoglycerate kinase

3. Phosphoglycerate mutase

4. Enolase

5. Pyruvate Kinase

Which enzyme performs substrate-level phosphorylation to form ATP

phosphoglycerate kinase

Which is the only redox enzyme in glycolysis

Glyceraldehyde-3-phosphate dehydrogenase

What electron carrier is generated by GAPDH

NADH

What molecule must be regenerated for glycolysis to continue

NAD+

Conversion of GAP to 1,3-BPG by GAPDH Summary

NAD+ is required as an electron acceptor in the active site of GAPDH to create a molecule with high phosphoryl-transfer potential (1,3-BPG) which can be used to generate ATP in a subsequent step

Which is not an irreversible reaction of glycolysis

Glyceraldehyde-3-phosphate dehydrogenase

What happens to pyruvate under aerobic conditions in humans

it forms acetyl-CoA

gluconeogenesis is the synthesis of

glucose from non-carbohydrate precursors

which non carbohydrate molecule can be used for gluconeogensis

Lactate, Alanine, Glycerol

What is the energy cost associated with gluconeogenesis for one glucose molecule

4 ATP + 2 GTP

Order of gluconeogenesis reaction

1. Pyruvate carboxylase

2. PEP carboxykinase

3. FBPase1

4. Glucose-6-phosphatase

Which of the following enzymes is found in both gluconeogenesis and glycolysis

phosphoglucoisomerase

What step of glycolysis is known as the committed step

fructose 6-phosphate to fructose 1,6-biphosphate

Why are glycolysis and gluconeogenesis reciprocally regulated

to prevent a futile cycle

Reciprocal Regulation of Glycolysis and Gluconeogenesis through allosteric effectors

Allosteric regulation of the above enzymes ensures when energy is needed or glycolytic intermediates are needed for biosynthesis, glycolysis will predominate. When there is a surplus of energy and glucose precursors, gluconeogenesis will take over

What is the main function of fructose 2,6-biphosphate

Activates glycolysis, inhibits gluconeogenesis

For phosphofructokinase-1 (PFK-1)

Low ATP stimulates, and fructose-2,6-biphosphate activates

Reciprocal Regulation of Glycolysis and Gluconeogenesis through hormone signaling

Insulin and glucagon signaling contribute to the pathways by regulating PFK2/FBPase2, which regulates [F-2,6-BP] in the cell

Which enzymes are stimulated by insulin signaling

PFK1 and PFK2

All of the enzymes of gluconeogenesis may be found in the cytosol EXCEPT ___, which is only found in the mitochondria

pyruvate carboxylase

Which enzyme complex links glycolysis to the citric acid cycle

Pyruvate dehydrogenase: pyruvate produced by glycolysis is converted into acetyl CoA, the fuel of the citric acid cycle

Pyruvate metabolism through the citric acid cycle and electron transport chain is an

aerobic process

What happens to pyruvate under anaerobic conditions in humans

It is converted to lactate, occurs to reoxidize NADH/regenerate NAD+ to sustain glycolysis in animal muscle cells

Niacin vitamin

Single six-membered ring with amide group branching

Pantothenate vitamin

Long, flexible chain containing amide bonds and central carbon with two methyl groups and a hydroxyl group

Riboflavin

Three-ring system with a straight-chain sugar alcohol extending downward from it

A reducer __ an electron __ an oxidizer, causing the reducer to become ___ while the oxidizer becomes ___

donates

to

oxidized

reduced

The coenzyme nicotinamide adenine dinucleotide (NAD+) is ___ to form ___ and goes on to interact ___ with its associated enzymes called

reduced

NADH

non-covalently

dehydrogenase

Coenzyme A becomes activated by ___ binding an ___ group via ___ bond, in which the fewest number of carbons it can carry is ___

covalently

acyl

thioester

2