Quiz #4/5
Quiz #4/5
• #4: Glycolysis (Tuesday, Feb 20th)
• #5: TCA cycle (Monday, Mar 5th)
• Pathways are in the books
• Quiz will have the entire pathway:
– All cofactors will be present
– Random intermediate and enzymes removed
• You fill in the missing names
– Draw the structure for 1 intermediate
• Indicated by a larger box
Enzyme Regulation
Conditions Affecting
Enzyme Activity
pH
temperature
pH
Effects of pH on Enzyme Activity
• Protonation state of side chains
– Variation in protein structure
– Substrate binding
– catalysis
• Ionization of substrate
– Substrate binding
Temperature
a
b
Protein
unfolding
Relative
Activity
Temperature
Control of Enzyme Availability
Principles of Genetic Regulation
Types of Enzymes
“Control
of Gene Expression”
• Constitutive Enzymes: e.g. glycolytic
enzymes and gluconeogenic enzymes
• Inducible Enzymes: e.g. b-galactosidase
• Repressible Enzymes: e.g. ten enzymes
of histidine biosynthesis
Negative Regulators
[Bind to operators or upstream repression sequences (URS)]
Inducer
–
O
Regulator
(Repressor)
Complex
Corepressor
O
–
Regulator
(Aporepressor)
Complex
(Repressor)
Inducible
e.g. lactose operon
Repressible
e.g. trp operon
Positive Regulators
[Bind to promoters, enhancers or upstream
activation sequences (UAS)]
Inducer
+
O
Regulator
+
Complex
"Activator"
Corepressor
Regulator
"Activator"
O
Complex
Inducible
e.g. cAMP
Repressible
e.g. nit-2
Regulation of Enzyme
Catalytic Activity
Covalent Modification
Allosteric Enzymes
Principles Governing Controls of
Enzyme Catalytic Activity
• Regulatory Enzymes
– Enzyme catalyzing committed, rate-limiting
step (often first step)
– Thermodynamically highly favorable reaction
• Outcomes of Regulation
– Feedback inhibition (fbi) of biosynthetic
pathways
– Modulation of metabolic flux
Reversible Covalent Modification
Protein Modification
(Phosphorylation/Dephosphorylation)
Page 390
Non-covalent Modification
Effectors or Ligands
Positive: activators
Negative: inhibitors
Allosteric Enzymes
(Modulation of Enzyme Catalytic Activity)
• Substrate Binding
• Catalytic Rate
• Both
Allosteric (Regulatory) Enzymes
(Inactive)
T
Substrates
Activators
Inhibitor
R (Active)
Homotropic Effects
M—M (Simple
Enzyme)
VO
negative cooperativity
positive cooperativity
[S]
Heterotropic Effects
no effector
Vo
negative effector
positive effector
[S]
Glycogen Phosphorylase
Figure 12-16
Regulation of Biosynthetic
Pathways
Rationale for Regulation
Biosynthesis
Macromolecules
Central
Metabolite
Product
(e.g. Amino Acid)
Catabolism
Nutrient
Efficiency and Flexibility
Biological Efficiency
• Biosynthesis
– Synthesize precursors not available in diet
– Cease synthesis when precursors become
available in diet (pre-existing enzymes)
– Produce precursors and macromolecules at
appropriate rates
• Catabolism
– Degrade most appropriate nutrients at
appropriate rates
Biological Flexibility
• Adaptaton to Dietary Changes
– Need for biosynthetic products
– Catabolism of new nutrients
– Control of pre-existing enzymes
• Metabolic Flux
– Rates of metabolism reflecting needs
for energy and macromolecular synthesis
Competing Reactions: Regulation
A
Enzyme 1
B
Enzyme 2
C
Control Mechanisms
• Control of Enzyme Availability
– Induction/repression
• Control of Enzyme Activity
– Covalent/Non-covalent
• Control of Substrate Availability
Types of Regulation
• Specific: pathway’s 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
Biosynthetic Pathways
ATP
Central
Metabolite
ADP + Pi
Product
(Amino Acid)
Simple Feedback Inhibition
Central
Metabolite
Product
(Amino Acid)
X
ATP
ADP + Pi
Complex Feedback Inhibition
Central
Metabolite
X
X
X
Product 1
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
Cumulative Feedback Inhibition
D
A
B
E
C
A
F
A
B
B
G
D
E
F
G
C
D
E
F
G
C
Concerted Feedback Inhibition
D
A
B
E
C
A
F
A
B
B
G
D
E
F
G
C
D
E
F
G
C
Isozymes
D
A
B
E
C
A
F
A
B
B
G
D
E
F
G
C
D
E
F
G
C
Modulation of Metabolic Flux
Energy Charge
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
Energy Charge
Anabolic pathways
(Biosynthesis)
Catabolic Pathways
(Degradation)
• Require ATP
• Produce ATP
• Activated
• Activated
– High EC (ATP)
• Inhibited
– Low EC (AMP)
– Low EC (AMP)
• Inhibited
– Hig EC (ATP)