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Enzyme mechanisms
Andy Howard
Introductory Biochemistry
2 November 2010
Biochem: Enzyme Mechanisms
11/02/2010
More about mechanisms
Many enzymatic mechanisms
involve either covalent catalysis or
acid-base interactions
 We’ll give some examples of several
mechanistic approaches
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Mechanism Topics
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Enzyme dynamics
Enzyme chemistry
Transition-state
binding
Diffusion-controlled
Reactions
Binding Modes of
Catalysis
Redox reactions
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Induced fit
Ionic intermediates
Active-site amino
acids
Serine proteases
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Reaction
How they illustrate
what we’ve learned
Specificity
Evolution
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The protein moves as well!
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Changes to active-site conformation:
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Help with substrate binding
Position the catalytic groups
Induce formation of a near-attack
conformation (NAC)
Help to break or make bonds
Facilitate conversion of S to P
Sometimes involve networks of
concerted amino acid changes
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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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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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Diffusion-controlled reactions
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Some enzymes are so efficient that the limiting
factor in completion of the reaction is diffusion of
the substrates into the active site:
These are diffusion-controlled reactions.
Ultra-high turnover rates: kcat ~ 109 s-1.
We can describe kcat / Km as catalytic efficiency
(or the specificity constant) of an enzyme. A
diffusion-controlled reaction will have a catalytic
efficiency on the order of 108 M-1s-1.
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Induced fit
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Refinement on original Emil Fischer lockand-key notion:
both the substrate (or transition-state)
and the enzyme have flexibility
Binding induces conformational changes
Cartoon
courtesy
Wikibooks.org
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Ionic reactions
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Define them as reactions that involve
charged, or at least polar, intermediates
Typically 2 reactants
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Electron rich (nucleophilic) reactant
Electron poor (electrophilic) reactant
Conventional to describe reaction as
attack of nucleophile on electrophile
Drawn with nucleophile donating
electron(s) to electrophile
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Attack on Acyl Group
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Transfer of an acyl group: section 14.6
Nucleophile Y attacks carbonyl carbon,
forming tetrahedral intermediate
X- is leaving group
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Direct Displacement
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Attacking group adds to face of
atom opposite to leaving group
Transition state can have five
ligands;
This is inherently less stable than
other attacks, but it can still work
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Cleavage Reactions
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Both electrons stay with one atom
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Covalent bond produces carbanion:
R3—C—H  R3—C:- + H+
Covalent bond produces carbocation:
R3—C—H  R3—C+ + :H-
One electron stays with each product
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Both end up as radicals
R1O—OR2  R1O• + •OR2
Radicals are highly reactive—
some more than others
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Cleavages by base
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Simple cleavage:
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—X—H + :B  —X:- + H—B+
This works if X=N,O; sometimes C
 Removal of proton from H2O to cleave C-X:
O
O
O —C—N  —C—OH + HN
—C—N
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HO
O
H
H :B
H—B+
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:B
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Cleavage by acid
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Covalent bond may break more easily if
one of its atoms is protonated
Formation of unstable intermediate,
R-OH2+, accelerates the reaction
Example:
R+
+
R—OH  R—OH2+
(Slow)
OH-

(Fast)
 R+ + H2O
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Low-barrier H-bonds
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Ordinary H-bonds buy us 10-30 kJ mol-1
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O—O separation = 0.28nm (similar for O-N)
O—H = 0.1 nm so H…O distance is 0.18nm
As the O’s get closer to each other, the bond
order gets closer to 0.5 for both
We than have an O-O distance ~ 0.22 nm &
much stronger (60 kJ mol-1) interaction
pKa for the two heteroatoms must be nearly
equal for this to happen
Several mechanisms employ these
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Oxidation-Reduction
Reactions
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Commonplace in biochemistry: EC 1
Oxidation is a loss of electrons
Reduction is the gain of electrons
In practice, often:
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oxidation is decrease in # of C-H bonds;
reduction is increase in # of C-H bonds
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Redox,
continued
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Intermediate electron acceptors and
donors are organic moieties or metals
Ultimate electron acceptor in aerobic
organisms is usually dioxygen (O2)
Anaerobic organisms usually employ
other electron acceptors
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Biological redox reactions
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Generally 2-electron transformations
Often involve alcohols, aldehydes, ketones,
carboxylic acids, C=C bonds:
R1R2CH-OH + X  R1R2C=O + XH2
R1HC=O + X + OH- R1COO- + XH2
X is usually NAD, NADP, FAD, FMN
A few biological redox systems involve metal ions
or Fe-S complexes
Usually reduced compounds are higher-energy
than the corresponding oxidized compounds
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One-electron
redox reactions
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FMN, FAD, some metal ions can be
oxidized or reduced one electron at a
time
With organic cofactors this generally
leaves a free radical in each of two
places
Subsequent reactions get us back to an
even number of electrons
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Covalent catalysis
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Reactive side-chain can be a
nucleophile or an electrophile, but
nucleophile is more common
 A—X + E  X—E + A
 X—E + B  B—X + E
Example: sucrose phosphorylase
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Net reaction:
Sucrose + Pi  Glucose 1-P + fructose
Fructose=A, Glucose=X, Phosphate=B
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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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Reactive sidechains in a.a.’s
AA
Group
Charge
@pH=7
Asp —COO-1
Glu —COO-1
His Imidazole ~0
Cys —CH2SH ~0
Tyr Phenol
0
Lys NH3+
+1
Arg guanidinium +1
Ser —CH2OH 0
Functions
Cation binding, H+ transfer
Same as above
Proton transfer
Covalent binding of acyl gps
H-bonding to ligands
Anion binding, H+ transfer
Anion binding
See cys
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Generalizations about activesite amino acids
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Typical enzyme has 2-6 key catalytic
residues
His, asp, arg, glu, lys account for 64%
Remember:
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pKa values in proteins sometimes different
from those of isolated aa’s
Frequency overall  Frequency in catalysis
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Rates often depend on pH
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If an amino acid that is necessary to
the mechanism changes protonation
state at a particular pH, then the
reaction may be allowed or
disallowed depending on pH
Two ionizable residues means there
may be a narrow pH optimum for
catalysis
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Papain as an example
1
relative reaction rate
Papain pH-rate profile
Cys-25
His-159
0
2
3
4
5
6
7
8
9
10
11
pH
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iClicker quiz, question 1
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
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
 (e) None of them would bind at all.
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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 amino
acids at position 1, 2, -1, . . .
Others are more promiscuous
O
CH
NH
R1
NH
C
O
C
NH
CH
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R-1
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Mechanism
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Active-site serine —OH …
Without neighboring amino acids, it’s fairly
unreactive
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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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
Primary differences are in P1 side chain
pocket, but that isn’t inevitable
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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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iClicker question #3
Which of the following serine
proteases would you expect to be
the most similar to human
pancreatic elastase?
 (a) Subtilisin from Bacillus subtilis
 (b) human neutrophil elastase
 (c) pig pancreatic elastase
 (d) human pancreatic chymotrypsin
 (e) they all would be equally similar.
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