Enzyme Mechanisms
and Regulation
Andy Howard
Introductory Biochemistry, Fall 2008
Tuesday 28 October 2008
Biochemistry: Mechanisms
1
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How do enzymes reduce
activation energies?
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We can illustrate mechanistic
principles by looking at specific
examples; we can also recognize
enyzme regulation when we see it.
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Mechanism Topics
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Mechanisms
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Induced-fit
Tight Binding of
Ionic
Intermediates
Serine proteases
Other proteases
Lysozyme
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Regulation
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Thermodynamics
Enzyme availability
Allostery, revisited
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Examining enzyme
mechanisms will help us
understand catalysis
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Examining general principles of
catalytic activity and looking at
specific cases will facilitate our
appreciation of all enzymes.
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Binding modes:
proximity
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We describe enzymatic mechanisms in terms
of the binding modes of the substrates (or,
more properly, the transition-state species) to
the enzyme.
One of these involves the proximity effect,
in which two (or more) substrates are
directed down potential-energy gradients to
positions where they are close to one
another. Thus the enzyme is able to defeat
the entropic difficulty of bringing substrates
together.
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Biochemistry: Mechanisms
William
Jencks
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Binding modes: efficient
transition-state binding
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Transition state fits even better
(geometrically and electrostatically) in
the active site than the substrate would.
This improved fit lowers the energy of
the transition-state system relative to
the substrate.
Best competitive inhibitors of an
enzyme are those that resemble the
transition state rather than the substrate
or product.
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Proline racemase
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Pyrrole-2-carboyxlate resembles
planar transition state
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Yeast aldolase
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Phosphoglycolohydroxamate binds
much like the transition state to the
catalytic Zn2+
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Adenosine deaminase with
transition-state analog
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Transition-state analog:
Ki~10-8 * substrate Km
Wilson et al (1991) Science 252: 1278
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
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ADA transition-state analog
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1,6 hydrate of
purine
ribonucleoside
binds with KI ~
3*10-13 M
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Induced fit
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Refinement on original Emil
Fischer lock-and-key notion:
both the substrate (or transitionstate) and the enzyme have
flexibility
Binding induces conformational
changes
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Example: hexokinase
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Glucose + ATP  Glucose-6-P + ADP
Risk: unproductive reaction with water
Enzyme exists in open & closed forms
Glucose induces conversion to closed
form; water can’t do that
Energy expended moving to closed form
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Hexokinase structure
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Diagram courtesy E. Marcotte, UT Austin
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Tight binding of ionic
intermediates
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Quasi-stable ionic species strongly bound
by ion-pair and H-bond interactions
Similar to notion that transition states are
the most tightly bound species, but these
are more stable
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Serine protease mechanism
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Only detailed mechanism that we’ll ask
you to memorize
One of the first to be elucidated
Well studied structurally
Illustrates many other mechanisms
Instance of convergent and divergent
evolution
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The reaction
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Hydrolytic cleavage of peptide bond
Enzyme usually works on esters too
Found in eukaryotic digestive enzymes and
in bacterial systems
Widely-varying substrate specificities
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Some proteases are highly specific for
particular aas at position 1, 2, -1, . . .
Others are more promiscuous
CH
NH
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R1
NH
C
C
CH
NH
O
R-1
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Mechanisms
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Mechanism
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Active-site serine —OH …
Without neighboring amino acids, it’s fairly
non-reactive
becomes powerful nucleophile because OH
proton lies near unprotonated N of His
This N can abstract the hydrogen at nearneutral pH
Resulting + charge on His is stabilized by its
proximity to a nearby carboxylate group on
an aspartate side-chain.
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Catalytic triad
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The catalytic triad of asp, his, and ser is
found in an approximately linear
arrangement in all the serine proteases,
all the way from non-specific, secreted
bacterial proteases to highly regulated
and highly specific mammalian
proteases.
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Diagram of first three steps
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Diagram of last four steps
Diagrams courtesy
University of Virginia
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Chymotrypsin as example
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Catalytic Ser is Ser195
Asp is 102, His is 57
Note symmetry of mechanism:
steps read similarly L R and R  L
Diagram courtesy of Anthony
Serianni, University of Notre Dame
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Oxyanion hole
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When his-57 accepts proton from Ser-195:
it creates an R—O- ion on Ser sidechain
In reality the Ser O immediately becomes
covalently bonded to substrate carbonyl carbon,
moving - charge to the carbonyl O.
Oxyanion is on the substrate's oxygen
Oxyanion stabilized by additional interaction in
addition to the protonated his 57:
main-chain NH group from gly 193 H-bonds to
oxygen atom (or ion) from the substrate,
further stabilizing the ion.
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Oxyanion
hole cartoon
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Cartoon courtesy Henry
Jakubowski, College of
St.Benedict / St.John’s
University
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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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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!
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Why would the nonproductive hexokinase
reaction H2O + ATP -> ADP + Pi
be considered nonproductive?
(a) Because it needlessly soaks up water
(b) Because the enzyme undergoes a wasteful
conformational change
(c) Because the energy in the high-energy
phosphate bond is unavailable for other
purposes
(d) Because ADP is poisonous
(e) None of the above
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iClicker quiz, question 2:
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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Question 3: what would bind
tightest in the TIM active site?
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(a) DHAP (substrate)
(b) D-glyceraldehyde (product)
(c) 2-phosphoglycolate
(Transition-state analog)
(d) They would all bind equally well
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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-
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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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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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