Transcript Enzyme Mechanisms

Enzymes V:
Specific Mechanisms;
Regulation
Andy Howard
Introductory Biochemistry
10 November 2008
Biochem: Specific Mechanisms
11/5/2009
Examples of mechanisms
We’ll look at the serine protease
mechanism in detail, 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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Significance
Catalytic residues
Sequence of events
Chymotrypsin
Evolution
Other mechanisms
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Regulation by
thermodynamics
Enzyme availability
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Transcription
Degradation
Compartmentation
Allostery
Cysteinyl proteases
Lysozyme
TIM
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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
R1
NH
C
C
NH
CH
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Mechanism
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Active-site serine —OH …
Without neighboring amino acids, it’s fairly
non-reactive (naked ser-OH pKa ~ 14)
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 negative 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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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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Non-iClicker quiz, question 1
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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-3-P (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-
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
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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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