Transcript Enzyme Mechanisms - Illinois Institute of Technology

Enzyme Mechanisms
and Regulation
Andy Howard
Introductory Biochemistry, Fall 2008
Tuesday 28 October 2008
Biochemistry: Mechanisms
1
10/28/2008
How do enzymes reduce
activation energies?

We can illustrate mechanistic
principles by looking at specific
examples; we can also recognize
enyzme regulation when we see it.
10/28/2008
Biochemistry: Mechanisms
p. 2 of 56
Mechanism Topics

Mechanisms





Induced-fit
Tight Binding of
Ionic
Intermediates
Serine proteases
Other proteases
Lysozyme
10/28/2008

Regulation



Thermodynamics
Enzyme availability
Allostery, revisited
Biochemistry: Mechanisms
p. 3 of 56
Examining enzyme
mechanisms will help us
understand catalysis

Examining general principles of
catalytic activity and looking at
specific cases will facilitate our
appreciation of all enzymes.
10/28/2008
Biochemistry: Mechanisms
p. 4 of 56
Binding modes:
proximity


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.
10/28/2008
Biochemistry: Mechanisms
William
Jencks
p. 5 of 56
Binding modes: efficient
transition-state binding


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.
10/28/2008
Biochemistry: Mechanisms
p. 6 of 56
Proline racemase

Pyrrole-2-carboyxlate resembles
planar transition state
10/28/2008
Biochemistry: Mechanisms
p. 7 of 56
Yeast aldolase

Phosphoglycolohydroxamate binds
much like the transition state to the
catalytic Zn2+
10/28/2008
Biochemistry: Mechanisms
p. 8 of 56
Adenosine deaminase with
transition-state analog


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.
10/28/2008
Biochemistry: Mechanisms
p. 9 of 56
ADA transition-state analog

1,6 hydrate of
purine
ribonucleoside
binds with KI ~
3*10-13 M
10/28/2008
Biochemistry: Mechanisms
p. 10 of 56
Induced fit



Refinement on original Emil
Fischer lock-and-key notion:
both the substrate (or transitionstate) and the enzyme have
flexibility
Binding induces conformational
changes
10/28/2008
Biochemistry: Mechanisms
p. 11 of 56
Example: hexokinase





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
10/28/2008
Biochemistry: Mechanisms
p. 12 of 56
Hexokinase structure

Diagram courtesy E. Marcotte, UT Austin
10/28/2008
Biochemistry: Mechanisms
p. 13 of 56
Tight binding of ionic
intermediates


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
10/28/2008
Biochemistry: Mechanisms
p. 14 of 56
Serine protease mechanism





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
10/28/2008
Biochemistry: Mechanisms
p. 15 of 56
The reaction




Hydrolytic cleavage of peptide bond
Enzyme usually works on esters too
Found in eukaryotic digestive enzymes and
in bacterial systems
Widely-varying substrate specificities


Some proteases are highly specific for
particular aas at position 1, 2, -1, . . .
Others are more promiscuous
CH
NH
10/28/2008
R1
NH
C
C
CH
NH
O
R-1
Biochemistry:
Mechanisms
p. 16 of 56
Mechanism




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.
10/28/2008
Biochemistry: Mechanisms
p. 17 of 56
Catalytic triad

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.
10/28/2008
Biochemistry: Mechanisms
p. 18 of 56
Diagram of first three steps
10/28/2008
Biochemistry: Mechanisms
p. 19 of 56
Diagram of last four steps
Diagrams courtesy
University of Virginia
10/28/2008
Biochemistry: Mechanisms
p. 20 of 56
Chymotrypsin as example



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
10/28/2008
Biochemistry: Mechanisms
p. 21 of 56
Oxyanion hole




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.
10/28/2008
Biochemistry: Mechanisms
p. 22 of 56
Oxyanion
hole cartoon

Cartoon courtesy Henry
Jakubowski, College of
St.Benedict / St.John’s
University
10/28/2008
Biochemistry: Mechanisms
p. 23 of 56
Modes of catalysis in serine
proteases




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
10/28/2008
Biochemistry: Mechanisms
p. 24 of 56
Specificity





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.
10/28/2008
Biochemistry: Mechanisms
p. 25 of 56
Chymotrypsin active site


Comfortably
accommodates
aromatics at S1 site
Differs from other
mammalian serine
proteases in specificity
Diagram courtesy School of
Crystallography, Birkbeck
College
10/28/2008
Biochemistry: Mechanisms
p. 26 of 56
Divergent evolution


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
10/28/2008
Biochemistry: Mechanisms
p. 27 of 56
iClicker quiz!






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
10/28/2008
Biochemistry: Mechanisms
p. 28 of 56
iClicker quiz, question 2:
Why are proteases often
synthesized as zymogens?




(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
10/28/2008
Biochemistry: Mechanisms
p. 29 of 56
Question 3: what would bind
tightest in the TIM active site?




(a) DHAP (substrate)
(b) D-glyceraldehyde (product)
(c) 2-phosphoglycolate
(Transition-state analog)
(d) They would all bind equally well
10/28/2008
Biochemistry: Mechanisms
p. 30 of 56
Convergent evolution


Reappearance of ser-his-asp triad in
unrelated settings
Subtilisin: externals very different from
mammalian serine proteases; triad same
10/28/2008
Biochemistry: Mechanisms
p. 31 of 56
Subtilisin mutagenesis



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.
10/28/2008
Biochemistry: Mechanisms
p. 32 of 56
Cysteinyl proteases



Ancestrally related to ser
proteases?
Cathepsins, caspases,
papain
Contrasts:



Cys —SH is more basic
than ser —OH
Residue is less hydrophilic
S- is a weaker nucleophile
than O-
10/28/2008
Diagram courtesy of
Mariusz Jaskolski,
U. Poznan
Biochemistry: Mechanisms
p. 33 of 56
Papain active site
Diagram courtesy
Martin Harrison,
Manchester University
10/28/2008
Biochemistry: Mechanisms
p. 34 of 56
Hen egg-white
lysozyme



Antibacterial protectant of
growing chick embryo
Hydrolyzes bacterial cell-wall
peptidoglycans
“hydrogen atom of structural biology”




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)
10/28/2008
Biochemistry: Mechanisms
p. 35 of 56
Mechanism of
lysozyme



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)
10/28/2008
Biochemistry: Mechanisms
p. 36 of 56
The
controversy
10/28/2008
Biochemistry: Mechanisms
p. 37 of 56
Regulation of enzymes


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:





Thermodynamics
Enzyme availability
Allostery
Post-translational modification
Protein-protein interactions
10/28/2008
Biochemistry: Mechanisms
p. 38 of 56
Thermodynamics as a
regulatory force



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
10/28/2008
Biochemistry: Mechanisms
p. 39 of 56
Enzyme availability



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?



Transcription (and translation)
Protein processing (degradation)
Compartmentalization
10/28/2008
Biochemistry: Mechanisms
p. 40 of 56
Transcriptional control



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



Proteins can bind to their own gene
Small molecules can bind to gene
Promoters can be turned on or off
10/28/2008
Biochemistry: Mechanisms
p. 41 of 56
Protein
degradation




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
10/28/2008
Biochemistry: Mechanisms
p. 42 of 56
Compartmentalization



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
10/28/2008
Biochemistry: Mechanisms
p. 43 of 56
Allostery




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
10/28/2008
Biochemistry: Mechanisms
p. 44 of 56
Substrates as allosteric
effectors (homotropic)



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
10/28/2008
Biochemistry: Mechanisms
p. 45 of 56
Other allosteric effectors
(heterotropic)



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
10/28/2008
Biochemistry: Mechanisms
p. 46 of 56
Cyclic AMP-dependent
protein kinases



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
10/28/2008
Biochemistry: Mechanisms
p. 47 of 56
Kinetics of
allosteric enzymes



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
10/28/2008
Biochemistry: Mechanisms
p. 48 of 56
T  R State transitions




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
10/28/2008
Biochemistry: Mechanisms
p. 49 of 56