Regulation of Metabolism

Rationale for Regulation

Biosynthesis
Macromolecules
Central
Metabolite

Product
(e.g. Amino Acid)

Catabolism

Nutrient

Biological Efficiency
Flexibility: adaptaton to dietary
changes
Need for biosynthetic products
Control of pre-existing enzymes

Modulation: biosynthesis only as fast

as needs for macromolecular syntesis

Competing Reactions: Regulation

A
Enzyme 1

Enzyme 2

Kinetic Controls

V1 =

V1
V2

k1[E1 ][S1 ]
Km1 + [S1]

V2 =

k2[E 2][S2]
Km2 + [S2]

k1

E1

S1

Km2 + [S2]

k2

E2

S2

Km1 + [S1 ]

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

Review of Genetic Regulation

General Principles

Types of Enzymes
Constitutive Enzymes: e.g. glycolytic
enzymes and gluconeogenic enzymes
Inducible Enzymes: e.g. b-galactosidase
Repressible Enzymes: e.g. ten enzymes
of histidine biosynthesis

Sites of Regulation
DNA

Transcription
initiation
polymerization
termination

RNA

Processing
splicing
capping
tailing
Translocation

cRNA

Translation
initiation
polymerization
termination

Proteins

Also
Turnover of RNAs and Proteins
Processing of precursor proteins

Prokaryotes: usually at transcription initiation.

Eukaryotes: can be anywhere!

Types of Regulation
Specific: one pathways substrate or
product
General: needs for C or N sources or
growth rates (e.g. energy charge)

Signals Mediating Regulation

Availability of
Substrates or Products
(Ligands)

Regulatory Proteins

Gene Organization and Control

Property

Prokaryotes

Eukaryotes

Coordinate

Coordinate

Operons

Dispersed

Magnitude

Large

Small

Complexity

Simple???

Complex

Coupled

Uncoupled

Regulation
Organization

Transcription
& Translation

Gene Expression in Bacteria

(Operon Model)
RNAP

Transcription

R2

Translation
P,O

Attenuation Signal
R1

Stop Codon
(Nonsense)

Upstream Regulatory Sequences

Promoter (general term)
UAS (Upstream Activation Sequence)
Enhancers
URS (Upstream Repression Sequence)
Operator

Binding of RNA Polymerase to

Promoter
Affected by regulators
Affected by strength of promoter:
provides appropriate variation in enzyme
levels

Gene Expression in Eukaryotes

Dispersed Genes

Mechanisms of Gene Regulation

Pathway

Terminology

Ligand

Regulator

Catabolic

Induction

Substrate

Negative (lac operon)

Positive (ara operon)

Anabolic

Repression

Product

Negative (trp operon)

Positive (amino acids in yeast)

Negative Regulators

[Bind to operators or upstream repression sequences (URS)]

Inducer

Regulator
(Repressor)

Complex

Inducible
e.g. lactose operon

Corepressor

Regulator
(Aporepressor)

Complex
(Repressor)

Repressible
e.g. trp operon

Positive Regulators

[Bind to promoters, enhancers or upstream

activation sequences (UAS)]

Inducer

Regulator

Complex
"Activator"

Inducible
e.g. cAMP

Corepressor

Regulator
"Activator"

Complex

Repressible
e.g. nit-2

Attenuation in Bacteria

(Coupled Transcription and Translation)

RNA polymerase
DNA

Protein
+

H3N

Ribosome

mRNA

Mechanism of Attenuation
"mRNA Leader"

5'

AUG

Upstream Open Reading Frame (uORF)

Stop

AUG

Gene(s)

trp codons

NOTE: Negative Regulatory System

Discovery of Attenuation
Charles Yanofsky

Control of Enzyme Activity

Irreversible Covalent Modification

Zymogen Activation
Proteolysis
Lysosomes
Proteosomes (ubiquitin)

Reversible Covalent Modification

(glucose)n

Pi

Phosphorylase
P

(glucose)n-1

glucose-1-P

Phosphorylase a
"active"
P

Phosphorylase
Phosphatase

4 H2 O

4 ADP

4 Pi

4 ATP

Phosphorylase
Kinase

Phosphorylase b
"inactive"

Non-covalent Modification
Effectors or Ligands

Negative Effectors

Active
Site

I
I

Regulatory
Site

"active"

"inactive"
or
less active

Positive Effectors

+
+

"inactive"
or
poorly active

"active"
or
more active

Allosteric Proteins

no effector
Vo

negative effector
positive effector
[S]

Energy Charge
(Daniel Atkinson)

Energy Charge =

1
2

2ATP + ADP
ATP + ADP + AMP

Steady-State E.C. = 0.93

ATP, ADP and AMP = Regulatory Ligands

Regulation of Degradative
Pathways

Degradative Pathways

Substrate

Central Metabolite
("Catabolite")

Enzyme Amount
Induction
(Inducer = Substrate)

Catabolite Repression

Lactose

b-Galactosidase

Galactose + Glucose

Negative Regulators

Inducer

Regulator
(Repressor)

Complex

Positive Regulators

Inducer

Regulator

Complex
"Activator"

Enzyme Activity
Regulation Unnecessary
No Substrate = No Flux

Lactose Utilization
E. coli
Lactose

b-galactosidase

Glucose + Galactose
Glycolysis
TCA Cycle

C Sources

NOTE: function is to provide carbon and

energy when substrate is available and
when products are needed.

Regulation of Enzyme Amount

Conditions

Enzyme Levels

Terminology

(C Source)

(b-galactosidase)

Glucose

~0.0

Uninduced
(Basal)

Lactose

1,000

Induction

Lactose +
Glucose

~0.0

Catabolite
Repression

Regulation
Specific Regulation: mediated by availability of
substrate called effector (or inducer) e.g.
lactose (allolactose) through its interaction with a
regulatory protein.
General Regulation: e.g. catabolite repression
analogous to repression in that endproduct
effector (catabolite co-repressor) prevents gene
expression, often by interacting with a regulatory
protein, but may use second messenger system
e.g. cAMP.

Physiological Manifestations of
Catabolite Repression
cells
Diauxic
Lag

log[cells]

b-galactosidase

b-galactosidase
Induction

Time
Use glucose
exclusively

Use lactose

Structure of Lac Operon

Structural Genes
P

Regulation of the Lac Operon

Structural G enes
Stru ctural Genes
RN AP

CA P

CAP
SITE

RNA Polym era se

cAM P

lactose

CAP = cata bolite

a ctiva tor
p rotein

Requirements for Gene Expression

Availability of Substrate: Lactose (or

allolactose)

and
Need for Product: low [glucose) > cAMP

Mechanism of Catabolite Repression

Adenylate
Cyclase

PPi + cAMP
ATP
Activation
Pyruvate
PEP
COOH
C
CH 2

OP

EI~P
EI

HPr
HPr~P

Soluble
Cytoplasmic
Proteins
(Common)

EIIIg ~P
EIIIg
Glucose-6-P

EIIg

Glucose

Inducible Operon
(Positive Regulator)

RNAP

o
Amino Acid

UAS

Binding of amino acid is required

to activate positive transcripiton
factor (regulator)

Hut Operon of Klebsiella aerogenes

Pathway

Regulation
hisR+ Enzyme Levels

hisRC Enzyme Levels

C Source

N Source

His

+His

His

+His

Glucose

NH3

Glucose

Limit NH3

105

100

100

Limit Glc

NH3

120

100

100

Limit Glc

Limit NH3

120

100

100

Mechanism of Regulation
Stru ctural Gen es
RN AP

CAP

R2

R1

cAMP

low gln

P,O

his

either one

Carbon Catabolite Repression

Nitrogen Metabolite Repression

Regulation of Biosynthetic
Pathways

Biosynthetic Pathways

ATP
Central
Metabolite

ADP + Pi
Product
(Amino Acid)

Enzyme Amount
Repression
Endproduct = Corepressor

Negative Regulator

Corepressor

Regulator
(Aporepressor)

Complex
(Repressor)

Positive Regulators
Corepressor

Regulator
"Activator"

Complex

Enzyme Activity
Feedback Inhibition
Endproduct = Ligand or Effector

Simple Feedback Inhibition

Central
Metabolite

Product
(Amino Acid)

X
ATP

ADP + Pi

Complex Feedback Inhibition

Central
Metabolite

Product 1

X
Product 2

Mechanisms of Complex Feedback

Inhibition
Cumulative: sum of individual inhibitions
Concerted: both end products required for
inhibition
Isoenzyme: two enzymes, each inhibitable
by different end product
Sequential: inhibition by accumulating
intermediate

Amino Acid Biosynthetic Operon

Positive Regulator

Pathway

Central
Metabolite

Amino
Acid

Regulation
Transcription
Translation
R

RNAP

UAS

Start Codon

+
Amino Acid

Stop Codon
(Nonsense)

Eukaryotes versus Prokaryotes

Properties
Increased Size: reduced membrane
surface to volume ratio
Increased Complexity: limited
solvent capacity
Uncoupled Transcription and
Translation: slower gene expression

Evolutionary Response
Organelles
Constitutive Enzymes

Problems
Intracellular Metabolite Transport
Competing Pathways

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)