Transcript Enzyme Mechanisms - Illinois Institute of Technology

Enzymes V:
Specific Mechanisms;
Regulation
Andy Howard
Introductory Biochemistry
4 November 2010
Enzyme Mechanisms & Regulation
11/4/2010
Examples of mechanisms
We’ll finish our detailed look at the
serine protease mechanism, and
then explore a few other
mechanisms to illustrate specific
ideas
 Then we’ll begin our discussion of
regulation of enzymes
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Mechanisms and Regulation
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Serine Proteases
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Cysteinyl proteases
Lysozyme
TIM
Regulation by
thermodynamics
Enzyme availability
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Chymotrypsin
Evolution
Other mechanisms
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Allostery
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Transcription
Degradation
Compartmentation
Mechanisms
Kinetics
PTM
Hemoglobin &
myoglobin as instances
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Modes of catalysis in serine
proteases
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Proximity effect: gathering of reactants in steps
1 and 4
Acid-base catalysis at histidine in steps 2 and 4
Covalent catalysis on serine hydroxymethyl
group in steps 2-5
So both chemical (acid-base & covalent) and
binding modes (proximity & transition-state) are
used in this mechanism
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What mechanistic concepts do
serine proteases not illustrate?
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Quaternary structural effects
(We’ll discuss this under regulation…)
Protein-protein interactions
(Becoming increasingly important)
Allostery
(also will be discussed under regulation)
Noncompetitive inhibition
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Specificity
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Active site catalytic triad is nearly invariant for
eukaryotic serine proteases
Remainder of cavity where reaction occurs
varies significantly from protease to protease.
In chymotrypsin  hydrophobic pocket just
upstream of the position where scissile bond sits
This accommodates large hydrophobic side
chain like that of phe, and doesn’t comfortably
accommodate hydrophilic or small side chain.
Thus specificity is conferred by the shape and
electrostatic character of the site.
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Chymotrypsin active site
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Comfortably
accommodates
aromatics at S1 site
Differs from other
mammalian serine
proteases in specificity
Diagram courtesy School of
Crystallography, Birkbeck
College
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Divergent evolution
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Ancestral eukaryotic serine proteases
presumably have differentiated into forms
with different side-chain specificities
Chymotrypsin is substantially conserved
within eukaryotes, but is distinctly
different from elastase
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iClicker quiz, question 1:
Why are proteases often
synthesized as zymogens?
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(a) Because the transcriptional machinery
cannot function otherwise
(b) To prevent the enzyme from cleaving
peptide bonds outside of its intended realm
(c) To exert control over the proteolytic reaction
(d) None of the above
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Convergent evolution
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Reappearance of ser-his-asp triad in
unrelated settings
Subtilisin: externals very different from
mammalian serine proteases; triad same
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Subtilisin mutagenesis
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Substitutions for any of the amino acids in the
catalytic triad has disastrous effects on the
catalytic activity, as measured by kcat.
Km affected only slightly, since the structure of
the binding pocket is not altered very much by
conservative mutations.
An interesting (and somewhat non-intuitive)
result is that even these "broken" enzymes
still catalyze the hydrolysis of some test
substrates at much higher rates than buffer
alone would provide. I would encourage you
to think about why that might be true.
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Cysteinyl proteases
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Ancestrally related to ser
proteases?
Cathepsins, caspases,
papain
Contrasts:
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Cys —SH is more basic
than ser —OH
Residue is less hydrophilic
S- is a weaker nucleophile
than O-
Diagram courtesy of
Mariusz Jaskolski,
U. Poznan
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Papain active site
Diagram courtesy
Martin Harrison,
Manchester University
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Hen egg-white
lysozyme
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Antibacterial protectant of
growing chick embryo
Hydrolyzes bacterial cell-wall
peptidoglycans
“hydrogen atom of structural biology”
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
HEWL
PDB 2vb1
0.65Å
15 kDa
Commercially available in pure form
Easy to crystallize and do structure work
Available in multiple crystal forms
Mechanism is surprisingly complex (14.7)
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Mechanism of
lysozyme
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Strain-induced destabilization of
substrate makes the substrate look more
like the transition state
Long arguments about the nature of the
intermediates
Accepted answer: covalent intermediate
between D52 and glycosyl C1 (14.39B)
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The
controversy
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Triosephosphate isomerase
(TIM)
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dihydroxyacetone phosphate 
glyceraldehyde-3-phosphate
Glyc-3-P
DHAP

Km=10µM
kcat=4000s-1
kcat/Km=4*108M-1s-1
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TIM mechanism
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DHAP carbonyl H-bonds to neutral imidazole
of his-95; proton moves from C1 to
carboxylate of glu165
Enediolate intermediate (C—O- on C2)
Imidazolate (negative!) form of his95 interacts
with C1—O-H)
glu165 donates proton back to C2
See Fort’s treatment
(http://chemistry.umeche.maine.edu/
CHY431/Enzyme3.html)
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Enzymes are under
several levels of control
Some controls operate at the
level of enzyme availability
 Other controls are exerted by
thermodynamics, inhibition, or
allostery
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Regulation of enzymes
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The very catalytic proficiency for which
enzymes have evolved means that their
activity must not be allowed to run amok
Activity is regulated in many ways:
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Thermodynamics
Enzyme availability
Allostery
Post-translational modification
Protein-protein interactions
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Thermodynamics as a
regulatory force
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Remember that Go’ is not the
determiner of spontaneity: G is.
Therefore: local product and substrate
concentrations determine whether the
enzyme is catalyzing reversible
reactions to the left or to the right
Rule of thumb: Go’ < -20 kJ mol-1 is
irreversible
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Enzyme availability
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The enzyme has to be where the
reactants are in order for it to act
Even a highly proficient enzyme has to
have a nonzero concentration
How can the cell control [E]tot?
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Transcription (and translation)
Protein processing (degradation)
Compartmentalization
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Transcriptional control
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mRNAs have short lifetimes
Therefore once a protein is degraded, it
will be replaced and available only if new
transcriptional activity for that protein
occurs
 Many types of transcriptional effectors
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Proteins can bind to their own gene
Small molecules can bind to gene
Promoters can be turned on or off
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Protein
degradation
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All proteins have
finite half-lives;
Enzymes’ lifetimes often shorter than
structural or transport proteins
Degraded by slings & arrows of outrageous
fortune; or
Activity of the proteasome, a molecular
machine that tags proteins for degradation
and then accomplishes it
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Compartmentalization
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If the enzyme is in one compartment and
the substrate in another, it won’t catalyze
anything
Several mitochondrial catabolic enzyme
act on substrates produced in the
cytoplasm; these require elaborate
transport mechanisms to move them in
Therefore, control of the transporters
confers control over the enzymatic system
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Allostery
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Remember we defined this as an effect on
protein activity in which binding of a ligand
to a protein induces a conformational
change that modifies the protein’s activity
Ligand may be the same molecule as the
substrate or it may be a different one
Ligand may bind to the same subunit or a
different one
These effects happen to non-enzymatic
proteins as well as enzymes
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Substrates as allosteric
effectors (homotropic)
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Standard example: binding of O2 to one
subunit of tetrameric hemoglobin induces
conformational change that facilitates
binding of 2nd (& 3rd & 4th) O2’s
So the first oxygen is an allosteric
effector of the activity in the other
subunits
Effect can be inhibitory or accelerative
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Other allosteric effectors
(heterotropic)
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Covalent modification of an enzyme by
phosphate or other PTM molecules can
turn it on or off
Usually catabolic enzymes are stimulated
by phosphorylation and anabolic
enzymes are turned off, but not always
Phosphatases catalyze
dephosphorylation; these have the
opposite effects
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Cyclic AMP-dependent
protein kinases
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Enzymes phosphorylate proteins with S or T
within sequence R(R/K)X(S*/T*)
Intrasteric control:
regulatory subunit or domain has a sequence
that looks like the target sequence; this binds
and inactivates the kinase’s catalytic subunit
When regulatory subunits binds cAMP, it
releases from the catalytic subunit so it can
do its thing
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Kinetics of
allosteric enzymes
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Generally these don’t obey MichaelisMenten kinetics
Homotropic positive effectors produce
sigmoidal (S-shaped) kinetics curves
rather than hyperbolae
This reflects the fact that the binding of
the first substrate accelerates binding of
second and later ones
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T  R State transitions
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Many allosteric effectors influence the
equilibrium between two conformations
One is typically more rigid and inactive,
the other is more flexible and active
The rigid one is typically called the “tight”
or “T” state; the flexible one is called the
“relaxed” or “R” state
Allosteric effectors shift the equilibrium
toward R or toward T
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MWC model
for allostery
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Emphasizes symmetry
and symmetrybreaking in seeing how
subunit interactions
give rise to allostery
Can only explain
positive cooperativity
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Koshland (KNF) model
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Emphasizes conformational changes from
one state to another, induced by binding
of effector
Ligand binding and conformational
transitions are distinct steps
… so this is a sequential model for
allosteric transitions
Allows for negative cooperativity as well
as positive cooperativity
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Heterotropic effectors
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Post-translational modification
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We’ve already looked at phosphorylation
Proteolytic cleavage of the enzyme to
activate it is another common PTM mode
Some proteases cleave themselves
(auto-catalysis); in other cases there’s an
external protease involved
Blood-clotting cascade involves a series
of catalytic activations
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Zymogens
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As mentioned earlier, this is a term for an
inactive form of a protein produced at the
ribosome
Proteolytic post-translational processing
required for the zymogen to be converted
to its active form
Cleavage may happen intracellularly,
during secretion, or extracellularly
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Blood clotting
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Seven serine proteases in cascade
Final one (thrombin) converts fibrinogen
to fibrin, which can aggregate to form an
insoluble mat to prevent leakage
Two different pathways:
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Intrinsic: blood sees injury directly
Extrinsic: injured tissues release factors that
stimulate process
Come together at factor X
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Cascade
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Protein-protein interactions
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One major change in biochemistry in the last
20 years is the increasing emphasis on
protein-protein interactions in understanding
biological activities
Many proteins depend on exogenous
partners for modulating their activity up or
down
Example: cholera toxin’s enzymatic
component depends on interaction with
human protein ARF6
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Globins as aids to
understanding
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Myoglobin and hemoglobin are wellunderstood non-enzymatic proteins
whose properties help us understand
enzyme regulation
Hemoglobin is described as an “honorary
enzyme” in that it “catalyzes” the reaction
O2(lung)  O2 (peripheral tissues)
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Setting the stage for this story
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Myoglobin is a 16kDa monomeric O2-storage
protein found in peripheral tissues
Has Fe-containing prosthetic group called
heme; iron must be in Fe2+ state to bind O2
It yields up dioxygen to various oxygenrequiring processes, particularly oxidative
phosphorylation in mitochondria in rapidly
metabolizing tissues
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Why is myoglobin needed?
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Free heme will bind O2 nicely;
why not just rely on that?
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Protein has 3 functions:
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Immobilizes the heme group
Discourages oxidation of Fe2+ to Fe3+
Provides a pocket that oxygen can fit into
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Setting the stage II
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Hemoglobin (in vertebrates, at least) is a
tetrameric, 64 kDa transport protein that
carries oxygen from the lungs to
peripheral tissues
It also transports acidic CO2 the opposite
direction
Its allosteric properties are what we’ll
discuss
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Structure
determinations
Myoglobin & hemoglobin were the
first 2 proteins to have their 3-D
structures determined experimentally
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Myoglobin: Kendrew, 1958
Hemoglobin: Perutz, 1958
Most of the experimental tools that
crystallographers rely on were
developed for these structure
determinations
Nobel prizes for both, 1965
(small T!)
11/4/2010 Enzyme Mechanisms & Regulation
QuickTi me™ and a
decompressor
are needed to see thi s pi cture.
Photo courtesy
EMBL
QuickTime™ and a
decompressor
are needed to see this picture.
Photo courtesy
Oregon State
Library
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