Transcript Enzyme Regulatory Strategies

Enzyme Regulation
What you Need to Know
• Understand the different ways that enzyme activity is
regulated.
• What factors influence enzyme activity?
• General understanding of symmetry model versus
sequential model of allosteric regulation
• How do positive versus negative effectors affect plots
of enzyme velocity versus substrate concentration?
• What are general classes of protein kinases?
• How is cAMP protein kinase regulated?
• Understand the different ways that Glycogen
phosphorylase is regulated
What Factors Influence Enzymatic Activity?
• The availability of substrates and cofactors usually determines
how fast the reaction goes
• As product accumulates, the apparent rate of the enzymatic
reaction will decrease
• Genetic regulation of enzyme synthesis and decay determines the
amount of enzyme present at any moment
• Enzyme activity can be regulated allosterically (instantaneous
response)
• Enzyme activity can be regulated through covalent modification
(interconvertable enzymes) (response times of seconds or less)
– i.e. protein kinases (activate Ser, Thr, Tyr side chains)
• Zymogens (irreversible process), isozymes, and modulator
proteins may play a role
Transcription Regulation
• The amount of enzyme synthesized by a cell is
determined by transcription regulation.
– Induction
– Repression
• Genetic controls over enzyme levels have a
response time of minutes to hours or longer.
• Once synthesized, an enzyme can be degraded via
normal turnover of the protein or through specific
decay mechanisms that target the enzyme for
destruction (i.e. ubiquitinin pathway).
What Factors Influence Enzymatic Activity?
Enzyme regulation by reversible covalent modification.
What Factors Influence Enzymatic Activity?
Zymogens are inactive precursors
of enzymes. Typically, proteolytic
cleavage produces the active
enzyme.
Proinsulin is an 86-residue precursor
to insulin
The proteolytic activation of chymotrypsinogen
Proteolytic Enzymes of the Digestive Tract
The Cascade of Activation Steps Leading to Blood Clotting
The intrinsic and extrinsic pathways
converge at factor X, and the final
common pathway involves the
activation of thrombin (cleaves at ArgGly peptide) and its conversion of
fibrinogen into fibrin, which aggregates
into ordered filamentous arrays that
become crosslinked to form the clot.
Isozymes Are Enzymes With Slightly Different Subunits (structurally
similar but catalytically distinct)
Muscle becomes anaerobic: pyruvate
from glucose via glycolysis. Requires
LDH to regenerate NAD+ so glycolysis
can continue. (A4 best at regenerating
NAD+)
Heart is aerobic using
lactate as fuel converting it
to pyruvate; to fuel Citric
Acid Cycle (B4 inhibited by
excess pyruvate)
Isozymes of lactate
dehydrogenase (LDH).
What Are the General Features of Allosteric Regulation?
Action at "another site"
• Enzymes situated at key steps in metabolic
pathways are modulated by allosteric effectors
• These effectors are usually produced elsewhere
in the pathway
• Effectors may be feed-forward activators or
feedback inhibitors
– Sigmoid Kinetics ("S-shaped")
• Do not obey Michaelis-Menten eq.
• Suggest 2nd order or higher
relationship between v and [S]
v proportional to [S]n
– Explanation: Cooperative Binding
Sigmoid v versus [S] plot. The dotted line represents the
hyperbolic plot characteristic of normal Michaelis-Menten
kinetics.
Can Allosteric Regulation Be Explained by
Conformational Changes in Proteins?
• Monod, Wyman, Changeux (MWC) Model:
allosteric proteins can exist in two states: R
(relaxed) and T (taut)
• In this model, all the subunits of an oligomer
must be in the same state
• T state predominates in the absence of
substrate S
• S binds much tighter to R than to T and
substrate inhibitors bind only to the T state
The Symmetry Model for Allosteric Regulation is Based
on Two Conformational States for a Protein
Allosteric effects: A and I binding to R and T, respectively.
The Symmetry Model for Allosteric Regulation is Based on
Two Conformational States for a Protein
Allosteric effects: A and I binding to R and T, respectively.
The Symmetry Model for Allosteric Regulation is Based on
Two Conformational States for a Protein
Allosteric effects: A and I binding to R
and T, respectively.
More about the MWC model
• Cooperativity is achieved because S binding
increases the population of R, which increases
the sites available to S
• Ligands such as S are positive homotropic
effectors
• Molecules that influence the binding of
something other than themselves are
heterotropic effectors
The Sequential Model for Allosteric Regulation is Based on
Ligand-Induced Conformation Changes
• An alternative model – proposed by Koshland,
Nemethy, and Filmer (the KNF model): ligand binding
triggers a conformation change in a protein
• If the protein is oligomeric, ligand-induced
conformation changes in one subunit may lead to
conformation changes in adjacent subunits
• The KNF model explains negative cooperativity
• The KNF model is termed the sequential model
The Sequential Model for Allosteric Regulation is
Based on Ligand-Induced Conformation Changes
The Sequential Model for Allosteric Regulation is Based
on Ligand-Induced Conformation Changes
The KoshlandNemethy-Filmer
model. Theoretical
curves for the
binding of a ligand
to a protein having
four identical
subunits, each with
one binding site for
the ligand.
Novel Activation and Regulation
• Pro-enzyme: Inactive with conventional catalytic triad and
deformed substrate binding pocket.
• Resting enzyme: Inactive with mis-oriented catalytic triad and
fully formed substrate binding pocket.
• Active enzyme: Fully active, with conventional catalytic triad and
fully formed substrate binding pocket.
Factor D
Pro-enzyme
Activated Enzyme
Factor D
Trypsin
Covalent Modification Regulates the Activity of Enzymes
• Enzyme activity can be regulated through reversible
phosphorylation
– This is the most prominent form of covalent modification in
cellular regulation
• Phosphorylation is accomplished by protein kinases
– Each protein kinase targets specific proteins for phosphorylation
• Phosphoprotein phosphatases catalyze the reverse
reaction – removing phosphoryl groups from proteins
• Kinases and phosphatases themselves are targets of
regulation
Protein Kinases
• Protein kinases phosphorylate Ser, Thr, and Tyr residues in target
proteins
• Kinases typically recognize specific amino acid sequences in their
targets (i.e. PKA phosphorylated proteins having searing or
threonine residues within an R(R/K)X(S/T) target consensus
sequence
• In spite of this specificity, all kinases share a common catalytic
mechanism based on a conserved core kinase domain of about
260 residues
• Kinases are often regulated by intrasteric control, in which a
regulatory subunit (or domain) has a pseudosubstrate sequence
that mimics the target sequence, minus the phosphorylatable
residue (i.e. RRGAI)
– A is stericly similar to S but lacks a phosphorylatable OH
group.
Protein Kinases (a protein Superfamily)
Protein kinase A is shown
complexed with a
pseudosubstrate peptide
(orange).
This complex also includes ATP
(red) and two Mn2+ ions (yellow)
bound at the active site.
Protein Kinases
Phosphorylation is Not the Only Form of Covalent
Modification that Regulates Protein Function
Cyclic AMP-dependent protein kinase is composed of
catalytic and regulatory subunits
cyclic AMP-dependent protein kinase (also known as
protein kinase A (PKA) is a 150- to 170-kD R2C2 tetramer
in mammalian cells.
The two R (regulatory) subunits bind cAMP; cAMP
binding releases the R subunits from the C (catalytic)
subunits. C subunits are enzymatically active as
monomers.
Some Enzymes are Controlled by Both Allosteric Regulation
and Covalent Modification
• Glycogen phosphorylase (GP) is an example of the many
enzymes that are regulated both by allosteric controls and
by covalent modification
• GP cleaves glucose units from nonreducing ends of
glycogen (phosphorolysis reaction)
• This converts glycogen into readily usable fuel in the form
of glucose-1-phosphate
GP converts glycogen into glucose-1-phosphate
• Glycogen phosphorylase (GP) is regulated both by
allosteric controls and by covalent modification
Phosphoglucomutase converts glucose-1-P into the
glycolytic substrate, glucose-6-P
The phosphoglucomutase reaction.
The structure of glycogen phosphorylase
• Glycogen phosphorylase is a dimer of identical 842
residue subunits
• Each subunit contains an active site (at the center of the
subunit) and an allosteric effector site near the subunit
interface
• A regulatory phosphorylation site is located at Ser14 on
each subunit
• A glycogen-binding site exerts regulatory control
• Each subunit contributes a “tower helix” (residues 262 to
278) to the subunit-subunit interface
• In the dimer, the tower helices extend from their
respective subunits and pack against each other
The structure of glycogen phosphorylase
Glycogen Phosphorylase Activity is Regulated Allosterically
• Muscle glycogen phosphorylase shows cooperativity in
substrate binding
• ATP and glucose-6-P are allosteric inhibitors of glycogen
phosphorylase
• AMP is an allosteric activator of glycogen phosphorylase
• When ATP and glucose-6-P are abundant, glycogen
breakdown is inhibited
• When cellular energy reserves are low (i.e., high [AMP]
and low [ATP] and [G-6-P]) glycogen catabolism is
stimulated
Glycogen Phosphorylase Activity is Regulated Allosterically
v versus S curves for glycogen phosphorylase.
(a) The response to the concentration of the substrate
phosphate (Pi).
(b) ATP is a feedback inhibitor.
(c) AMP is a positive effector. It binds at the same site as ATP
providing reciprocal regulation.
Glycogen phosphorylase conforms to the MWC model
•
•
•
•
The active form of the enzyme is designated the R state
The inactive form of the enzyme is denoted the T state
AMP promotes the conversion to the active state
ATP, glucose-6-P, and caffeine favor conversion to the
inactive T state
• A significant conformation change occurs at the subunit
interface between the T and R state
• This conformational change at the interface is linked to a
structural change at the active site that affects catalysis
Glycogen Phosphorylase is Controlled by Both Allosteric
Regulation and Covalent Modification
The mechanism of covalent
modification and allosteric
regulation of glycogen
phosphorylase.
A Conformation Change Regulates Activity of
Glycogen Phosphorylase
The major conformational
change that occurs in the Nterminal residues upon
phosphorylation of Ser14. Ser14
is shown in red.
N-terminal conformation of
phosphorylated enzyme
(phosphorylase a): yellow.
N-terminal conformation of
unphosphorylated enzyme
(phosphorylase b): cyan.
Regulation of GP by Covalent Modification
• In 1956, Edwin Krebs and Edmond Fischer showed that a
‘converting enzyme’ could convert phosphorylase b to
phosphorylase a
• Three years later, Krebs and Fischer show that this
conversion involves covalent phosphorylation
• This phosphorylation is mediated by an enzyme cascade
Glycogen phosphoryase is activated by a cascade of reactions
The hormone-activated enzymatic cascade that leads to
activation of glycogen phosphorylase.
cAMP is a Second Messenger
• Cyclic AMP is the intracellular agent of
extracellular hormones - thus a ‘second
messenger’
• Hormone binding stimulates a GTP-binding
protein (G protein), releasing G(GTP)
• Binding of G(GTP) stimulates adenylyl cyclase
to make cAMP
Enzyme Cascade Regulates Phosphorylase Covalent Modification
Receptor
Cell Membrane
Gα (GTP)
Gα (GTP)
Adenylyl Cyclase
Hormone
cAMP
cAMP
G-protein (GDP)
GDP
GTP
active Glycogen Pa
2ADP 2ATP
ATP
cAMP
Inactive cAMP-DPK
P
P
active PK
Inactive Glycogen Pb
cAMP
ADP
ATP
Inactive PK
Active cAMP-DPK