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CHAPTER 10: REGULATORY STRATEGIES
Traffic signals control the
flow of traffic
INTRODUCTION
The activity of enzymes must often be regulated so that they
function at the proper time and place.
Enzymatic activity is regulated in five principal ways:
1. Allosteric Control
• Enzyme activity is controlled by the binding of small signal
molecules at regulatory sites
• Allosteric proteins show the property of cooperativity: activity
at one functional site affects the activity at others
• Aspartate transcarbamoylase (ATCase)
• Hemoglobin
CHAPTER 10
INTRODUCTION
2. Multiple Forms of Enzymes
• Isozymes are homologous enzymes within a single organism
- Catalyze the same reaction
- Slightly different in structure and catalytic/regulatory
properties
- Expressed in a distinct place or at a distinct stage of
development
CHAPTER 10
3. Reversible Covalent Modification
• Alters the catalytic properties of many enzymes
- Phosphorylation by protein kinases
- Dephosphorylation by protein phosphatases
- Protein kinase A
INTRODUCTION
4. Proteolytic Activation
• Activation of proenzymes (zymogens) by proteolytic
cleavage
- Chymotrypsin, trypsin, and pepsin are activated by this
mechanism
- Blood clotting is due to a cascade of zymogen activations
CHAPTER 10
5. Controlling the Amount of Enzyme Present
• Enzyme activity is regulated by adjusting the amount of
enzyme present
- This regulation usually takes place at the level of
transcription
10.1 ASPARTATE TRANSCARBAMOYLASE
The enzyme catalyzes the 1st step in the biosynthesis of
pyrimidines:
• The condensation of aspartate and carbamoyl phosphate
• The committed step in the pathway for pyrimidine nucleotide
such as CTP
CHAPTER 10
Fig 10.1 ATCase reaction.
10.1 ASPARTATE TRANSCARBAMOYLASE
It was found that ATCase is inhibited by CTP
Feedback inhibition
• The inhibition of an enzyme by the end product of the
pathway
CHAPTER 10
Fig 10.1 CTP inhibits ATCase.
Allosteric regulation
• CTP is structurally quite
different from the substrates
• CTP must bind to a site
distinct from the active site
SIGMOIDAL KINETICS
The dependence of the reaction rate on [Asp]
• Sigmoidal curve
• Cooperativity – the binding of substrate to one active site
increases the activity at the other active sites
10.1 ASPARTATE TRANSCARBAMOYLASE
Fig 10.3 ATCase displays
sigmoidal kinetics.
• Does not follow Michealis-Menten
kinetics
The majority of allosteric enzymes
display sigmoidal kinetics
• Cooperation between subunits in
hemoglobin
CATALYTIC AND REGULATORY SUBUNITS
ATCase can be separated into regulatory (r) and catalytic (c)
subunits by treatment with p-hydroxymercuribenzoate (p-HMB)
• Catalytic subunit (c3), three chains (34 kd each)
• Regulatory subunit (r2), two chains (17 kd each)
10.1 ASPARTATE TRANSCARBAMOYLASE
Fig 10.4 Modification of cysteine residues. Fig 10.5 Ultracentrifugation studies of ATCase.
Native p-HMB treated
CATALYTIC AND REGULATORY SUBUNITS
The larger subunit displays catalytic acitivity; unresponsive to
CTP, not sigmoidal kinetics – catalytic subunit
The smaller subunit can bind CTP; no catalytic acitivty –
regulatory subunit
The subunits combine rapidly when they are mixed
2 c3 + 3 r2 → c6r6
The reconstituted enzyme has the same structure and
allosteric/catalytic properties as those of the native enzyme
10.1 ASPARTATE TRANSCARBAMOYLASE
STRUCTURE OF ATCASE
Two catalytic trimers are stacked one on top of the other
Significant contacts between the catalytic and the regulatory
subunits
10.1 ASPARTATE TRANSCARBAMOYLASE
4-Cys bound
Fig 10.6 Structure of ATCase.
STRUCTURE OF ATCASE
A bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA)
was used for the ATCase-substrate analog complex structure
• PALA is a potent inhibitor for ATCase
10.1 ASPARTATE TRANSCARBAMOYLASE
Fig 10.7 PALA, a bisubstrate analog.
STRUCTURE OF ATCASE
The structure of the ATCase-PALA complex
• PALA binds at site lying at the boundaries between pairs of c
chains within a catalytic trimer
10.1 ASPARTATE TRANSCARBAMOYLASE
Fig 10.8 The active site of ATCase.
STRUCTURE OF ATCASE
Remarkable change in quaternary structure on PALA binding
ATCase shows two distinct quaternary forms: the T (tense)
state and the R (relaxed) state
10.1 ASPARTATE TRANSCARBAMOYLASE
Fig 10.9 The T-to-R state transition
in ATCase.
STRUCTURE OF ATCASE
How can we explain the enzyme’s sigmoidal kinetics?
In the absence of substrate, almost all the enzyme molecules
are in the T state
• Low affinity for substrate; low catalytic activity
The substrate binding to one active site
• Increases the likelihood that the entire enzyme shifts to the R
state with higher affinity
• Conversion of the enzyme into the R state causes more
substrate to bind to the active site
• Cooperativity
10.1 ASPARTATE TRANSCARBAMOYLASE
STRUCTURE OF ATCASE
The sigmoidal curve can be pictured as a composite of two
Michealis-Menten curves
The T-to-R transition is taking place within a narrow range of
substrate concentration
10.1 ASPARTATE TRANSCARBAMOYLASE
Fig 10.8 Basis for the sigmoidal curve.
• Makes it possible to respond
to small changes in substrate
concentration
CTP EFFECTS ON THE T-TO-R EQUILIBRIUM
CTP inhibits the action of ATCase
Structure of CTP-bound ATCase
• The enzyme is in the T state
• CTP binds to the regulatory domain
• The binding site is more than 50 Å from
the active site
10.1 ASPARTATE TRANSCARBAMOYLASE
Fig 10.11 CTP stabilizes the T state.
CTP EFFECTS ON THE T-TO-R EQUILIBRIUM
The binding of CTP shifts the equilibrium toward the T state
• Stabilizes the T state
• Decreases net enzyme activity
• Increases the initial phase of the sigmoidal curve
10.1 ASPARTATE TRANSCARBAMOYLASE
Fig 10.13 Effect of CTP on ATCase kinetics.
Fig 10.12 The R state and the T state are in equilibrium.
CTP EFFECTS ON THE T-TO-R EQUILIBRIUM
ATP increases the reaction rate at a given Asp concentration
• Competes with CTP for binding to regulatory sites
• High ATP means high purine: needs to make more pyrimidine
for balance
10.1 ASPARTATE TRANSCARBAMOYLASE
Fig 10.14 Effect of ATP on ATCase kinetics.
• High ATP means high energy
for mRNA synthesis and DNA
replication: leads to the
synthesis of more pyrimidines
http://www.youtube.com/watch?v=5aW0C3-IHVo
10.2 REGULATORY STRATEGIES IN ISOZYMES
Isozymes are enzymes that differ in AA sequence yet catalyze
the same reaction
Have different kinetic parameters or respond to different
regulatory molecules
Permits the fine-tuning of metabolism to meet the needs of a
given tissue or developmental stage
Lactate dehydrogenase
• Involved in glucose synthesis and metabolism
• Two isozymic peptides exist: H isozyme and M isozyme;
75% identical sequence
• Functions as a tetramer: many different combinations exist
CHAPTER 10
10.2 REGULATORY STRATEGIES IN ISOZYMES
The isozymes (H4 & M4) are functionally different
• H4: inhibited by high levels of pyruvate; functions in aerobic
environment
• M4: no inhibition by high levels of pyruvate; functions in
anaerobic environment
• H2M2 has intermediate properties
CHAPTER 10
Fig 10.16 Isozymes of lactate dehydrogenase.
The rat heart LDH isozyme profile
Days before (-) & after (+) birth
M4
H4
The tissue-specific forms of lactate dehydrogenase
in adult rat tissues
10.3 COVALENT MODIFICATION
CHAPTER 10
KINASES AND PHOSPHATASES
Phosphorylation is a regulatory mechanism used in every
metabolic process in eukaryotic cells
• 30% of eukaryotic proteins are phosphorylated
• Catalyzed by protein kinases
• Protein kinases are one of the largest protein families: more
than 500 protein kinases in human beings
• Fine-tuned regulation according to a specific tissue, time, or
substrate
• ATP is the most common donor
• Ser, Thr, and Tyr are the acceptors
10.3 COVALENT MODIFICATION
KINASES AND PHOSPHATASES
10.3 COVALENT MODIFICATION
KINASES AND PHOSPHATASES
Tyrosine kinases
• Play pivotal roles in growth regulation
• Mutations are observed in cancer cells
Ser/Thr kinases
10.3 COVALENT MODIFICATION
KINASES AND PHOSPHATASES
Substrate specificity of Ser/Thr kinases
• Some kinases phosphorylate a single protein or several
closely related ones
• Multifuncational kinases modify many different targets
- Recognize the consensus sequence, Arg-Arg-X-Ser/Thr-Z;
X a small residue; Z, a large hydrophobic residue
10.3 COVALENT MODIFICATION
KINASES AND PHOSPHATASES
Protein phosphatases remove
phosphoryl groups attached to proteins
• Turn off the signaling pathways
activated by kinases
• One class of conserved phosphatases
(PP2A) suppresses the cancer-
promoting activity of certain kinases
10.3 COVALENT MODIFICATION
Irreversible / unidirectional
Take place only by enzymes
PHOSPHORYLATION IS HIGHLY EFFECTIVE
Phosphorylation is a highly effective means of regulating the
activities of target proteins for several reasons:
1. The free energy of phosphorylation is large
• 20 ~ 30 kJ/mol (about 5 kcal/mol)
• A free energy change of 5.69 kJ/mol (1.36 kcal/mol)
corresponds to a factor of 10 in an equilibrium constant
• Phosphorylation shifts the equilibrium by 104
2. Adds two negative charges to a modified protein
• These charge can cause a large conformational change
• Such structural changes can markedly alter substrate binding
and catalytic activity
10.3 COVALENT MODIFICATION
PHOSPHORYLATION IS HIGHLY EFFECTIVE
Phosphorylation is a highly effective means of regulating the
activities of target proteins for several reasons:
3. A phosphoryl group can form three or more H-bonds
• Can make specific interaction
4. Phosphorylation and dephosphorylation can take place in less
than a second or over a span of hours
• The kinetics can be adjusted to meet the timing needs of a
physiological process
10.3 COVALENT MODIFICATION
PHOSPHORYLATION IS HIGHLY EFFECTIVE
Phosphorylation is a highly effective means of regulating the
activities of target proteins for several reasons:
5. The effects of phosphorylation can be highly amplified
• A single activated kinase can phosphorylate hundreds of
target proteins in a short interval
6. ATP is a cellular energy currency
• The use of ATP as a phosphoryl-group donor links the energy
status of the cell to the regulation of metabolism
10.3 COVALENT MODIFICATION
CYCLIC AMP ACTIVATES PROTEIN KINASE A
Cyclic AMP (cAMP) is an
intracellular messenger formed by
the cyclization of ATP
cAMP (>10 nM) activates a key
enzyme, protein kinase A (PKA)
The activated PKA alters the
activities of target proteins by
phosphorylation
Most effects of cAMP in eukaryotic
cells are achieved through the
activation of PKA
10.3 COVALENT MODIFICATION
PKA consists of two kinds of subunits:
• A 49-kD regulatory (R) subunit and a 38-kD catalytic (C) subunit
• In the absence of cAMP, PKA exists in a R2C2 form
The binding of cAMP to the R subunit relieve its inhibition of the C subunit
• Each R chain contains the sequence Arg-Arg-Gly-Ala-Ile
(pseudosubstrate sequence), which occupies the catalytic site of C
• The binding of cAMP allosterically removes the sequence resulting in
the activation of C
CYCLIC AMP ACTIVATES PROTEIN KINASE A10.3 COVALENT MODIFICATION
Fig 10.17 Regulation of PKA.
THE CRYSTAL STRUCTURE OF PKA
The X-ray crystal structure of PKA complexed with ATP and a
20-residue peptide inhibitor
10.3 COVALENT MODIFICATION
Two lobes: the smaller lobe,
contacts with ATP-Mg2+; the
larger lobe, binds to the peptide
substrate
Residues 40 to 280 are
conserved in all known protein
kinases
Fig 10.18 PKA bound to an inhibitor.
THE CRYSTAL STRUCTURE OF PKA
The bound peptide in the structure occupies the active site
10.3 COVALENT MODIFICATION
Two guanidinium groups
interact with three carboxylates
in the active site
Two Leu make a hydrophobic
site to accommodate the Ile of
the peptide
Fig 10.19 Binding of pseudosubstrate to protein kinase A.
The binding of cAMP allosterically
blocks this interaction resulting in
the activation of C
GLEEVEC
A tyrosine-kinase inhibitor used in the
treatment of multiple cancers, most
notably Philadelphia chromosome-
positive (Ph+) chronic myelogenous
leukemia (CML)
Received FDA approval in May 2001
Imatinib was one of the first cancer
therapies; a paradigm for research in
cancer therapeutics
Imatinib has been cited as the first of
the exceptionally expensive cancer
drugs
10.3 COVALENT MODIFICATION
Time magazine cover of 28
May 2001 detailing Glivec as a
'cure' for cancer.
10.4 REGULATION BY PROTEOLYTIC CLEAVAGE
Specific proteolysis is a common means of activating
enzymes
• The inactive precursor is called a zymogen or a proenzyme
Examples of regulation by proteolytic cleavage
1. Digestive enzymes
CHAPTER 10
10.4 REGULATION BY PROTEOLYTIC CLEAVAGE
Examples of regulation by proteolytic cleavage
2. Blood clotting – thrombin, cascade
3. Some protein hormones
• Insulin is derived from proinsulin by proteolytic cleavage
4. Developmental processes: conversion of procollagenase into
collagenase
• The metamorphosis of a tadpole into a frog: resorption of
large amounts of collagen from the tail
• Break down of collagen in a mammalian uterus after delivery
• The conversion is precisely timed in these remodeling
processes
CHAPTER 10
10.4 REGULATION BY PROTEOLYTIC CLEAVAGE
Examples of regulation by proteolytic cleavage
5. Apoptosis (programmed cell death)
• Eliminates damaged or infected cells and controls the
shapes of body parts in the course of development
• Apoptosis is mediated by caspases, proteolytic enzymes
• Caspases are generated from procaspases by proteolytic
cleavage
• Caspases function to cause cell death in most organisms
ranging from C. elegans to human beings
CHAPTER 10
CLEAVAGE OF CHYMOTRYPSINOGEN
Chymotrypsinogen
• Consisting of 245 AAs
• Synthesized in the
pancreas
• Inactive form of
chymotrypsin
• Cleaved by trypsin and
converted into two peptides
• Self-cleaved to produce
three peptide chains and
two dipeptides
10.4 REGULATION BY PROTEOLYTIC CLEAVAGE
Fig 10.21 Proteolytic activation of chymotrypsin.
HOW THE CLEAVAGE ACTIVATES THE ZYMOGEN? The cleavage generates a new interaction between the N-terminal
of Ile16 and the side chain of Asp194
10.4 REGULATION BY PROTEOLYTIC CLEAVAGE
The new interaction triggers a
number of conformational
changes
• Residues 187, 192, and 193
• The changes generates the
substrate-specificity site for
hydrophobic groups
• Generation of the oxyanion
hole
• Highly localized
conformational change Fig 10.22 Conformations of chymotrypsinogen
(red) and chymotrypsin (blue).
TRYPSIN IS THE COMMON ACTIVATOR
Trypsin is the common activator of all the pancreatic zymogens
• Trypsinogen, chymotrypsinogen, proelastase,
procarboxypeptidase, and prolipase
• The formation of trypsin by enteropeptidase is the master
activation step.
10.4 REGULATION BY PROTEOLYTIC CLEAVAGE
Fig 10.23 Zymogen activation by
proteolytic cleavage.
TRYPSIN IS THE COMMON ACTIVATOR
The activity of trypsin is controlled by a pancreatic trypsin inhibitor
• The inhibitor is a 6-kd protein
10.4 REGULATION BY PROTEOLYTIC CLEAVAGE
Fig 10.24 Interaction of trypsin with
its inhibitor.
The dissociation constant of the complex is 0.1 pM
• Substrate analog
• Preorganized structure
• Cleavage rate of the inhibitor by
trypsin is very slow (several
months of half-life)
BLOOD CLOTTING
Enzymatic cascades in biochemical systems
• Achieve a rapid response; an initial signal triggers a series of
steps each of which is catalyzed by an enzyme
• Activation of 10 enzymes by an enzyme can activate 104
enzymes in 4 steps
Blood clots are formed by a cascade of zymogen activations
• Small amounts of the initial factors suffice to trigger the cascade
10.4 REGULATION BY PROTEOLYTIC CLEAVAGE
BLOOD CLOTTING
10.4 REGULATION BY PROTEOLYTIC CLEAVAGE
Fig 10.26 Blood clotting cascade.
Blood clotting is achieved by the
interplay of the intrinsic, extrinsic,
and final common pathways
Begins with the activation of factor
XII by contact with abnormal
surfaces produced by injury
Triggered by trauma, which releases
tissue factor (TF)
TF forms a complex with VII, which
initiates a cascade-activating
thrombin
Inactive form, in red
Active form, in yellow
Activated by thrombin, with *
FLUORESCENT PROTEINS
History
Structure
Mechanism of the fluorescence
Mechanism of the various colors
Applications
Due: the day of the 1st exam (will be early April)
HOMEWORK #1
FLUORESCENT PROTEINS
HOMEWORK #1
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