Download Enzymes: Regulation 2-3

BIOC 460 Summer 2011
Enzymes: Regulation 2-3
Reversible
R
ibl covalent
l t modification
difi ti
Association with regulatory proteins
Irreversible covalent modification/proteolytic
cleavage
Reading: Berg, Tymoczko & Stryer, 6th ed.,
Chapter 10, pp. 283-299, Chapter 14, pp. 389-391
Problems: pp. 300-302, Chapter 10: #7, 10, 12, 13
•
•
•
Key Concepts
Activities of many key enzymes are regulated in cells, based on
metabolic needs/conditions in vivo.
Regulation of enzyme activity can increase or decrease substrate
binding affinity and/or kcat.
5 ways to regulate protein activity (including enzyme activity):
1. allosteric control
2
2.
multiple forms of enzymes (isozymes)
3. reversible covalent modification -- example:
• phosphorylation/dephosphorylation
• phosphorylation (phosphoryl transfer from ATP to specific -OH
group(s) on protein) catalyzed by protein kinases
• dephosphorylation (hydrolytic removal of the phosphate groups)
catalyzed by protein phosphatases
4
4.
interaction with regulatory proteins – examples:
• protein kinase A (PKA)
• Ca2+-calmodulin-dependent kinases
5. irreversible covalent modification, including proteolytic activation
(zymogen activation)
• examples:
– digestive proteases like chymotrypsin and trypsin
– blood clotting cascade
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Enzyme Regulation cont’d
3. Reversible Covalent Modification
• Modification of catalytic or other properties of proteins by covalent
attachment of a modifying group
• modification catalyzed by a specific enzyme.
• modifying group removed by a different enzyme
• Enzymes
E
can cycle
l between
b t
active
ti and
d inactive
i
ti (or
( more and
d less
l
active)
ti )
states by chemical modification.
•
•
•
allosteric regulation: an immediate and localized response, so rapid
activity changes
covalent modifications: slower and longer-lasting effects with
coordinated systemic effects
(e.g., a single hormone can trigger covalent modification events that
change activities of metabolic enzymes in a many tissues and cells.)
Activities of modifying/demodifying enzymes themselves are
regulated, allosterically (making process sensitive to changes in
concentration of small molecules that act as "signals"), or by another
reversible covalent modification process, or both.
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Phosphorylation/dephosphorylation
• probably the most common means of regulating enzymes, membrane
channels, virtually every metabolic process in eukaryotic cells
• Phosphorylation
– Kinases: catalyze phosphoryl transfer involving ATP (usually)
• named for molecule that "receives" phosphate group
• e.g., hexokinase transfers terminal phosphate from ATP to a variety
of hexose sugars like glucose (→ glucose-6-phosphate).
– General reaction catalyzed by kinases:
– (target) R-OH + ATP <==> R-OPO32– + ADP
– Protein kinases: kinases that transfer phosphoryl group from ATP to a
Ser-OH, Thr-OH, or Tyr-OH on a target protein)
• D
Dephosphorylation
h
h
l ti
– phosphate group removed by hydrolysis of phosphate ester (transfer
of phosphate to H2O)
– Dephosphorylation of enzymes is catalyzed by a specific PROTEIN
phosphatase.
Protein Kinases
• VERY important regulatory components in eukaryotic cells
• ⊗Go’ << 0 (equilibrium
lies far to right)
• Kinase reactions
essentially irreversible
• can’t make ATP
this way
Berg et al., p. 285
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•
•
•
2 classes of protein kinases:
1. Serine/Threonine protein kinases: recipient group on target protein is
a Ser-OH or Thr-OH.
2. Tyrosine kinases: recipient group on target protein is a Tyr-OH.
Recipient (target) protein's properties/conformation/activity altered by
phosphorylation
– Often, phosphorylation causes subtle conformational change that (if
target is an enzyme) increases or decreases catalytic activity
activity, or
– causes target to interact (or not to interact) with some other cellular
component.
Protein kinases themselves are regulated, often by allosteric effects of a
small signaling molecule, as in the examples below:
PROTEIN PHOSPHATASES
• catalyze hydrolysis of phosphate ester bonds in phosphorylated target
proteins = dephosphorylation
• Equilibrium lies far to the right -- irreversible in 55.5 M H2O.
• Dephosphorylation is NOT the reverse of protein kinase-catalyzed
phosphorylation reaction.
• Both types of reaction are irreversible.
• (Active) catalyst (kinase or phosphatase) needed for significant reaction
rates, so
• Cell's "decision" about what fraction of target protein is
phosphorylated vs. dephosphorylated depends on how active the
specific protein kinase is vs. how active the specific protein
phosphatase is.
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"Cycles" of phosphorylation/dephosphorylation hydrolyze ATP:
1. Target protein-OH + ATP → Target protein-OPO32– + ADP
2. Target protein-OPO32– + H2O → HOPO32– + Target protein-OH
3. Net reaction: ATP + H2O → ADP + HOPO32– (hydrolysis of ATP)
Standard free energy change, ⊗G°' = –31 kJ/mol, but
Actual free energy change under cellular conditions ⊗G' = ~ –50 kJ/mol.
• High negative free energy change makes
phosphorylation/dephosphorylation
cycle unidirectional in cell (essential for
a process whose rate is being regulated)
• 2 effects of large negative ⊗G' for
protein phosphorylation:
1. Some of net negative free energy
Change from phosphoryl transfer
makes reaction irreversible.
2. Some free energy is conserved in the phosphorylated protein -phosphorylation of even one site on a protein can shift
conformational equilibrium in protein structure by a large factor,
say 104.
• The 2 conformations can have very different catalytic or kinetic properties.
Biochemical Cascades: cellular/biochemical processes with
multiplicative effects
• Cascade: a series of events in which each event in series is catalyzed by
an enzyme activated in previous event.
– First event triggered by some signal that initiates cascade, e.g., a
hormone binding to a receptor, or a wound triggering the blood clotting
cascade
– Cascade produces rapid and enormous amplification of original
signal because every activated enzyme molecule can itself catalyze
conversion of many substrates (substrates often = other enzymes).
• Example: Suppose one signaling molecule triggers activation of one
molecule of Enzyme 1.
• Single molecule of active Enzyme 1 activates 100 molecules of Enzyme 2.
• Each of the 100 molecules of active Enzyme 2 activates 100 molecules of
Enzyme 3
3.
• Each of those 10,000 molecules of active Enzyme 3 activates 100
molecules of Enzyme 4.
• The 106 molecules of active Enzyme 4 each activates 100 molecules of
Enzyme 5 -- we're up to 100 million active Enzyme 5 molecules!
• Real cascades involve a lot more than 100 products per enzyme molecule,
with very rapid reactions, so geometric progression produces rapid and
enormous response.
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Adenylate Cascade and Protein Kinase
4. Interaction with regulatory proteins (Chapter 14, pp. 389-391)
Protein Kinase Cascades
• Phosphorylation as a control mechanism → highly amplified effects:
One single activated protein kinase molecule can phosphorylate
hundreds of target proteins in a very short time.
phosphorylation,
• If target proteins themselves are enzymes activated by phosphorylation
each activated enzyme then can carry out many, many catalytic cycles
on its substrate.
• Result of cascade: a major multiplicative effect between starting signal
(say, one small molecule binds to one protein kinase molecule to activate
it) and final outcome several steps away
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Protein Kinase Specificity
• Some protein kinases "multifunctional" -- phosphorylate many different
target proteins
• A particular kinase always phosphorylates a residue in a specific
sequence or a "consensus" sequence. (Sequences phosphorylated
by that kinase very similar but not all identical)
identical).
• example:
consensus sequence in all target proteins phosphorylated by
protein kinase A: Ser or Thr in this consensus sequence:
….–Arg–Arg–X–Ser–Z–… or …. –Arg–Arg–X–Thr–Z–…
X = a small amino acid residue; Z = a large hydrophobic residue
• Protein Kinase A binds other substrate protein sequences with a much
lower affinity
affinity, so doesn
doesn'tt phosphorylate them very often
often.
• Other protein kinases are very specific not only for local sequence but
also for 3-dimensional structure around it, and phosphorylate only a
single target protein or a small number of closely related target proteins.
Protein Kinase A
•
•
•
•
great example of integration of allosteric regulation and regulation by
reversible covalent modification (phosphorylation)
How does cAMP activate PKA?
cAMP binding alters quaternary structure of protein kinase A.
PKA inactive form (without cAMP bound): 2 catalytic subunits + 2 regulatory
subunits.
regulatory subunits inhibitory -- C2R2 quaternary form can't phosphorylate
targets.
cAMP binding to R subunits makes R’s dissociate from C subunits.
Berg et al., 5th ed., Fig. 10-28 (similar to 6th ed. Fig. 10-17)
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PKA: How does R binding keep C subunits inactive?
• Specific AA sequence in R subunit of PKA that binds to the C subunit is
actually a pseudosubstrate sequence:
• …. –Arg–Arg–Gly–Ala–Ile–…
Compare with consensus sequence where PKA phosphorylates targets:
….–Arg–Arg–X–Ser–Z–… or …. –Arg–Arg–X–Thr–Z–…
X = a small amino acid residue; Z = a large hydrophobic residue
• But R subunit sequence has Ala instead of Ser or Thr, so can't be
phosphorylated.
Knowing the sequence info above, to what part of PKA catalytic
subunits’ structure would regulatory subunits bind? (How would
that binding inhibit C subunit activity?)
Summary:
• cAMP binds to R subunits → conformational change, affects subunit interface.
reduces
d
bi
binding
di affinity
ffi it off R (inhibitory)
(i hibit ) subunits
b it ffor C subunits.
b it
• (cAMP)R-R(cAMP) complex dissociates from C subunits, releasing the 2 C’s
• Individual C subunits
active when free
Structure of protein kinase A catalytic subunit bound to Mg2+•ATP and
a 20-residue pseudosubstrate peptide inhibitor
(structure determined by X-ray crystallography)
Berg et al., 5th ed. ,
<--- Fig. 10-29
Berg et al.,6th ed.
Fig. 10-18 ---->
• ATP•Mg2+ + part of inhibitor bound in deep cleft between 2 "lobes" of protein,
ATP bound more to one lobe, inhibitor binding more to other lobe
• Substrate peptide binding → lobes move closer together
(conformational change/induced fit).
• Restricting domain closure used to regulate protein kinase activity.
• Essentially all known protein kinases have conserved same catalytic core,
residues 40-280 (out of 350 residues total) of PKA catalytic subunit
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Adenylate Cascade and Activation of Protein Kinase A by
cyclic AMP (cAMP)
1.
2.
3.
4.
5.
Regulatory cascade starts with hormone binding to extracellular
receptor → conformational changes in membrane proteins
Signal transduction (communication from one protein to another) →
activation of adenylate cyclase
adenylate cyclase: enzyme catalyzing intracellular production of
cyclic AMP (cAMP) by cyclization starting with ATP as substrate
cAMP: a small molecule (a nucleotide)
cAMP activates protein kinase A (PKA, aka "cAMP-dependent
protein kinase").
–
cAMP an important intracellular signaling molecule in both
prokaryotic and eukaryotic cells.
–
cAMP = a "second messenger": signaling molecule whose production
is under the control of other "messengers" such as hormones coming
to the cell from the extracellular environment
–
Primary role cAMP: activation of protein kinase A.
Active PKA then phosphorylates specific target proteins → many
different effects in the cell.
Major amplification effect of PKA activation: each activated molecule
of PKA can phosphorylate a LOT of molecules of target proteins.
The “adenylate cyclase cascade”
Berg et al., Fig. 2121-15
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Example 2: Ca2+-Calmodulin and CAM-Dependent Kinases
– Ca2+ a ubiquitous cytosolic messenger (signaling molecule).
– Ca2+ concentration "sensed" by Ca2+-binding proteins that
communicate signal to other proteins by protein-protein interactions.
Examples:
Calmodulin (CaM)
Troponin C (TnC, protein homologous to CaM in muscle cells,
regulating contraction in response to Ca2+)
– Calmodulin (CaM; Mr 17,000):
– example of a [Ca2+]-sensing protein
– changes conformation when it binds
Ca2+
– In
I Ca
C 2+-bound
b
d fform, CaM
C M bi
binds
d tto and
d
regulates activities of many CaMdependent proteins -- enzymes, pumps,
etc.
– Mode of binding of Ca2+ to calmodulin
Berg et al., Fig. 14-13 ---->
(CaM)
2+
Ca coordinated to 6 O atoms from
protein and 1 O atom from H2O (top)
CaM structure
• 4 high-affinity Ca2+ binding sites
• each site in an "EF hand" structural motif
– EF hand motif formed by
– Repeating Ca2+ binding motifs in
structure of CaM
helix-loop-helix unit
– CaM: 2 domains, each with 2 EF hand
– common Ca2+ binding
motif
Ca2+-binding motifs
– Ca2+ = green sphere
– 2 domains connected by
flexible 〈 helix
Berg et al., Fig. 14-15
Enzymes: Regulation 2-3
Berg et al., Fig. 3-25
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Conformational changes in calmodulin on calcium binding
• In absence of Ca2+, EF hands have hydrophobic cores buried inside the
protein.
• Binding of Ca2+ to each EF hand → structural changes that expose
hydrophobic patches on CaM surface.
• Hydrophobic patches serve as "docking regions" for binding target proteins.
• Central helix in CaM
– flexible even in the Ca2+-bound
state
– folds back on itself when the 2
Ca2+ domains of CaM bind to
(blocks access
of ATP to active
target proteins
• Target proteins all have a
positively charged,
charged amphipathic
〈-helix
• Ca2+-CaM binds to positively
charged, amphipathic 〈 helices
in the enzymes it regulates.
• CaM kinase peptide, purple
Berg et al., Fig. 14-16a
site in this
conformation)
(Target protein
activated by
Ca2+-CAM)
Ca2+-CaM binding to target enzyme’s amphipathic helix
stabilizes activated conformation of target enzyme.
• After Ca2+ binding (step 1), 2 halves of Ca2+-CaM clamp down around
target amphipathic helix in CaM Kinase I (step 2), binding it through
hydrophobic and ionic interactions.
• Result: "extraction" of C-terminal 〈 helix in CaM kinase I so it’s no longer
blocking active site → active conformation of CaM kinase I
(conformation that can bind ATP).
Berg et al., Fig. 14-16b
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5. Regulation of Enzyme Activity by Specific Proteolytic
Cleavage
• Some enzymes biosynthesized as catalytically inactive precursor
polypeptide chains
– Precursors fold in 3 dimensions
– Later activated by enzyme-catalyzed cleavage (hydrolysis) of 1 or
more specific peptide bonds
• ZYMOGENS (or proenzymes): inactive precursors
• zymogen activation: cleavage/activation process
Examples:
• 1) mammalian digestive enzymes
More examples of enzymes/proteins activated by specific
proteolysis
2) blood clotting: a cascade of proteolytic activations (→ rapid response,
with lots of amplification)
3)
some protein hormones synthesized as inactive precursors
–
e g insulin -- synthesized as proinsulin
e.g.,
–
final hormone generated by specific proteolysis to remove a peptide
4)
collagen
–
a fibrous protein (water-insoluble)
–
synthesized as procollagen, a water-soluble precursor
5) apoptosis (programmed cell death) mediated by caspases:
–
proteases synthesized as procaspases
–
activated by regulatory signals
6) many developmental processes controlled by precisely timed activation
of proenzymes
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1) digestive enzyme activation
• Chymotrypsin as example
• Zymogen = chymotrypsinogen
(Berg et al., Figs. 10-20 and 10-21)
(1st clip → activity)
Secretion of
zymogens by
pancreatic
acinar cells
(diffuse away)
Proteolytic activation of chymotrypsinogen:
• First cleavage (catalyzed by trypsin) between Lys15 and Ile16 generates
new 〈-amino group on Ile16
• Conformational change results: new N-terminus of larger product
chain (Ile16 residue) turns inward and makes new salt link that
stabilizes the active conformation of chymotrypsin:
Conformational change →
1 formation of substrate specificity site (hydrophobic pocket where
1.
"R1" specificity group of substrate binds)
2. "completion" of orientation of groups
to form oxyanion hole (for tight
binding of transition states in
acylation/deacylation mechanism.)
((1st clip
p → activity)
y)
Berg et al.,
Fig. 10-22
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Zymogenic Activation Cascade
The Importance of Control of Zymogen Activation
• Trypsin initiates activation of all the pancreatic zymogens.
– Enteropeptidase, enzyme secreted by cells that line the duodenum
(small intestine), activates a small amount of trypsinogen to trypsin,
which coordinates control of zymogen activation outside cells.
• What would happen if even a few zymogen molecules, especially
trypsinogen, were accidentally activated INSIDE the pancreatic
acinar cells?
• What prevents premature activation of pancreatic zymogens inside
cells?
•
Small, very specific, very tight-binding inhibitor proteins inside cell
inhibit any protease molecule that's accidentally prematurely
activated.
example: pancreatic trypsin inhibitor, PTI (6000 M.W.)
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• "PTI" binds VERY tightly to trypsin -- not even 8 M urea or 6 M
guanidine HCl dissociate the complex!
• Part of PTI binds in active site of trypsin, with a Lys residue of PTI
occupying R1 "specificity pocket".
• Pancreatic trypsin
inhibitor is a substrate,
but the peptide bond
"after" that Lys is
cleaved only VERY
slowly (time scale
of months).
• Combination of very
tight binding and
very slow catalytic
turnover makes
PTI a very effective
inhibitor.
Another tight-binding protease inhibitor helps prevent
emphysema.
• Emphysema results from loss of elasticity (elastic fibers and other
connective tissue proteins) in alveolar walls of the lungs, so CO2 can't be
exhaled effectively, so there isn't room for inhaling much fresh air (O2).
• Neutrophils (white blood cells that engulf invading bacteria) secrete
elastase.
• Excess elastase in blood plasma can hydrolyze elastic fibers in
alveolar walls of the lungs → emphysema.
• To prevent elastase from running amok in plasma, liver makes and
secretes a plasma protein, 〈1-antiproteinase (used to be called “alpha1antitrypsin”, but that’s a misnomer – it binds much tighter to elastase than
t trypsin).
to
t
i )
alpha1-antiproteinase in blood plasma keeps elastase inhibited,
protecting lungs from damage.
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Consequences of alpha1-antiproteinase deficiency
• Inherited disorders: defects either in its structure, making it less
effective as an inhibitor, or slowing down its secretion from liver and
thus reducing its concentration in plasma
1. genetic deficiency in 〈1-antiproteinase → increased
probability of developing emphysema.
emphysema
2. Cigarette smoke damages the inhibitor.
• Component of cigarette smoke oxidizes a Met residue in 〈1antiproteinase that's required for binding to elastase
(oxidation → non-functional inhibitor)
• Result: smokers continually inactivate 〈1-antiproteinase in
their lungs and thus are also much more likely to develop
emphysema.
• Imagine the results of a combination of a genetic deficiency and cigarette
smoke!
Learning Objectives
•
•
•
•
•
•
Terminology: cAMP, consensus sequence, pseudosubstrate, cascade,
reciprocal regulation, zymogen
Describe in general terms how cells carry out reversible covalent
modification of enzymes, and how the modification would be removed.
Name (“generic” names) the types of enzymes that catalyze
phosphorylation and dephosphorylation of proteins, specify what types of
amino acid functional groups are generally the targets of phosphorylation,
and show the structure of such an enzyme functional group before and
after phosphorylation.
Explain whether the dephosphorylation reaction is actually the chemical
reverse of the phosphorylation reaction, and if not, what type of reaction
the dephosphorylation represents.
Explain the regulation of protein kinase A (PKA) activity by cAMP,
including
g quaternary
q
y structural changes
g in PKA triggered
gg
by
y cAMP
binding. What is a "pseudosubstrate" and how does it relate to the role
of the regulatory subunits in PKA?
Briefly discuss the structure of calmodulin (± Ca2+), including structure of
the “EF hand” motif, and how Ca2+-calmodulin activates target proteins as
an example of how a regulatory protein works.
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Learning Objectives, continued
•
•
•
•
•
•
Describe the general mechanism by which zymogens are activated →
active enzymes.
Briefly describe the structural change that occurs upon the activation of
chymotrypsinogen, including what changes occur in the active site.
protective mechanism that keeps
p p
prematurely
y activated
Discuss the p
pancreatic digestive enzymes inside the acinar cells from autodigesting
the pancreas, and describe/name an example.
Give an example of a protease inhibitor that inhibits elastase.
Explain how a deficiency (or an inactivating chemical event) in 〈1antiproteinase (formerly called 〈1-antitrypsin) contributes to emphysema.
Explain how a cascade of catalysts (e.g., in PKA activation, or in blood
g) results in amplification
p
of a signal.
g
clotting)
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