(5) regulation

8/10/2019 (5) Regulation

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Regulation of Metabolism


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Biological Efficiency

Flexibility: adaptaton to dietarychanges

Need for biosynthetic products Control of pre-existing enzymes

Modulation: biosynthesis only as fast

as needs for macromolecular syntesis


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Competing Reactions: Regulation

A

B C

Enzyme 1 Enzyme 2


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Kinetic Controls

V1

V2

k1

k2

E1

E2

S1

S2

Km2+ [S2]

Km1+ [S1]

V1=k1[E1][S1]

Km1+ [S1]V2=

k2[E2][S2]

Km2+ [S2]

=


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Control Mechanisms

Control of Enzyme Amount Induction and Repression

Catabolite Repression Attenuaton

Control of Enzyme Activity Modulation of k or Vmax(rare)

Control of Kms Control of Substrate Availability


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Sites of Regulation

Transcription

initiation

polymerization

termination

Translation

initiation

polymerization

termination

DNA RNAProcessing

splicing

cappingtailing

Translocation

cRNA Proteins

Also

Turnover of RNAs and Proteins

Processing of precursor proteins

Prokaryotes: usually at transcription initiation.

Eukaryotes: can be anywhere!


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Types of Regulation

Specific: one pathways substrate orproduct

General: needs for C or N sources orgrowth rates (e.g. energy charge)


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Signals Mediating Regulation

Availability of

Substratesor Products(Ligands)

Regulatory Proteins


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Gene Organization and Control

Property Prokaryotes Eukaryotes

Regulation Coordinate Coordinate

Organization Operons Dispersed

Magnitude Large Small

Complexity Simple??? ComplexTranscription& Translation

Coupled Uncoupled


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Gene Expression in Bacteria

(Operon Model)RNAP

R2

A B C D

R1

P,O L

Transcription

Translation

Attenuation SignalStop Codon(Nonsense)


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Upstream Regulatory Sequences

Promoter (general term)

UAS(Upstream Activation Sequence) Enhancers

URS(Upstream Repression Sequence) Operator


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Binding of RNA Polymerase to

Promoter

Affected by regulators Affected by strength of promoter:

provides appropriate variation in enzymelevels


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Gene Expression in Eukaryotes

Dispersed Genes


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Mechanisms of Gene Regulation

Pathway Terminology Ligand RegulatorCatabolic Induction Substrate Negative (lacoperon)

Positive (araoperon)

Anabolic Repression Product Negative (trp operon)

Positive (amino acids in yeast)


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Negative Regulators[Bind to operatorsor upstream repression sequences (URS)]

O

O

Inducer

Inducible

e.g. lactose operon

Regulator(Repressor)

Complex

Corepressor

Regulator(Aporepressor)

Complex(Repressor)

Repressible

e.g. trp operon


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Positive Regulators[Bind to promoters, enhancersor upstream

activation sequences (UAS)]

O

O +

Inducer

Inducible

e.g. cAMP

Regulator Complex"Activator"

+

Corepressor

Regulator"Activator"

Complex

Repressible

e.g. nit-2


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Attenuation in Bacteria(Coupled Transcription and Translation)

DNA

mRNA

Protein

RNA polymerase

Ribosome+

H3N


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Mechanism of Attenuation

A B C

5'

"mRNA Leader"

AUG AUGStop Gene(s)

trp codons

Upstream Open Reading Frame (uORF)

NOTE: Negative Regulatory System


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Discovery of Attenuation

Charles Yanofsky


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Control of Enzyme Activity


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Irreversible Covalent Modification

Zymogen Activation

Proteolysis Lysosomes

Proteosomes (ubiquitin)


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Reversible Covalent Modification

PP

PP

4 ATP

4 ADP4 H2O

Phosphorylase a "active"

Phosphorylase b "inactive"

+

Phosphorylase

Phosphatase

Phosphorylase

Kinase4 Pi

(glucose)n-1 + glucose-1-PPhosphorylase

(glucose)n + Pi


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Non-covalent Modification

Effectors or Ligands


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Negative Effectors

"active"

Regulatory Site

Active Site

"inactive" orless active

I

I

+


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Positive Effectors

+

"active" ormore active

"inactive" or

poorly active

++


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Allosteric Proteins

positive effector

negative effector

no effecto

[S]

Vo


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Energy Charge

(Daniel Atkinson)

Steady-State E.C. = 0.93

ATP, ADP and AMP = Regulatory Ligands

Energy Charge 12

2ATP + ADPATP + ADP + AMP

=


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Regulation of DegradativePathways


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Degradative Pathways

Central Metabolite("Catabolite")

Substrate


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Enzyme Amount

Induction(Inducer = Substrate)

Catabolite Repression

b-GalactosidaseLactose Galactose + Glucose


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Negative Regulators

O

Inducer

Regulator

(Repressor)

Complex


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Positive Regulators

O +

Inducer

Regulator Complex"Activator"


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Enzyme Activity

Regulation Unnecessary

No Substrate = No Flux


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Lactose Utilization

Lactose Glucose + Galactoseb-galactosidase

E. coli

GlycolysisTCA Cycle

C Source

NOTE: function is to provide carbon andenergy when substrate is available andwhen products are needed.


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Regulation of Enzyme Amount

Conditions(C Source)

Enzyme Levels(b-galactosidase)

Terminology

Glucose ~0.0 Uninduced(Basal)

Lactose 1,000 Induction

Lactose +

Glucose

~0.0 Catabolite

Repression


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Regulation

Specific Regulation: mediated by availability ofsubstrate called effector (or inducer) e.g.lactose (allolactose) through its interaction with a

regulatory protein. General Regulation: e.g. catabolite repressionanalogous to repression in that endproducteffector (catabolite co-repressor) prevents geneexpression, often by interacting with a regulatory

protein, but may use second messenger system e.g. cAMP.


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Physiological Manifestations ofCatabolite Repression

log[cells]

b-galactosidase

cells

b-galactosidase

DiauxicLag

Induction

Use glucoseexclusively

Use lactose

Time


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Structure of LacOperon

Z

Structural Genes

Y AOP


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RNAP CAP R P O Y A

Stru ctur al Genes

CAPSITE

Z

RNA Polymerase

Structu ral Gen es

cAMP lactose

CAP = catab oliteactivator

p rotein

Regulation of the LacOperon


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Requirements for Gene Expression

Availability of Substrate: Lactose (or

allolactose)

and Need for Product: low [glucose) > cAMP


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Mechanism of Catabolite Repression

ATP

HPr

COOH

C

CH2

OP

PEP

Pyruvate

EI

EI~P

HPr~P EIIIg

EIIIg~P

Glucose

Glucose-6-P

PPi+ cAMP

AdenylateCyclase

Activation

EIIg

SolubleCytoplasmic

Proteins(Common)


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Inducible Operon(Positive Regulator)

+

o

A B C DRNAPR

UAS L

Amino Acid

P

Binding of amino acid is required

to activate positive transcripiton

factor (regulator)


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HutOperon of Klebsiella aerogenes


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Pathway


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Regulation

C Source N Source His +His His +His

Glucose NH3 0 0 0 0

Glucose Limit NH3 0 105 100 100

Limit Glc NH3 0 120 100 100

Limit Glc Limit NH3 0 120 100 100

hisR+Enzyme Levels hisRCEnzyme Levels


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Mechanism of Regulation

RN AP R 2CAP R 1 A B C D

Structural Genes

cAMP low gln

P,O

his

either one

Carbon Catabolite Repression

Nitrogen Metabolite Repression


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Regulation of BiosyntheticPathways


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Biosynthetic Pathways

ATP

CentralMetabolite

Product(Amino Acid)

ADP + Pi


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Enzyme Amount

Repression

Endproduct = Corepressor


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Negative Regulator

O

Corepressor

Regulator(Aporepressor)

Complex(Repressor)


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Positive Regulators

O+

Corepressor

Regulator"Activator"

Complex


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Enzyme Activity

Feedback Inhibition

Endproduct = Ligand or Effector


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Simple Feedback Inhibition

X

ATP

CentralMetabolite

Product(Amino Acid)

ADP + Pi


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Complex Feedback Inhibition

CentralMetabolite

Product 1

Product 2

XX

X


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Mechanisms of Complex FeedbackInhibition

Cumulative: sum of individual inhibitions

Concerted: both end products required forinhibition

Isoenzyme: two enzymes, each inhibitableby different end product

Sequential: inhibition by accumulatingintermediate


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Amino Acid Biosynthetic Operon

Positive Regulator


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Pathway

CentralMetabolite

Amino

Acid


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Regulation

+

o

A B C DRNAPR

UAS L

Transcription

Translation

Stop Codon(Nonsense)

Amino Acid

Start Codon

P


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Eukaryotes versusProkaryotes


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Properties

Increased Size: reduced membranesurface to volume ratio

Increased Complexity: limitedsolvent capacity

Uncoupled Transcription and

Translation: slower gene expression


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Evolutionary Response

Organelles

Constitutive Enzymes


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Problems

Intracellular Metabolite Transport

Competing Pathways


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Regulatory Solutions

Separate Metabolic Pathways Different intermediates Different enzymes (control of enzyme

activities) Physical Separation of Metabolic Pathways

Location Multienzyme Complexes

(Control of Substrate Availability)

(5) regulation

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