Transcript Enzyme Mechanisms - Illinois Institute of Technology

Enzyme Mechanisms
Andy Howard
Introductory Biochemistry, Fall 2008
Thursday 23 October 2008
Biochemistry: Enzyme Mechanisms
1
10/23/2008
How do enzymes reduce
activation energies?
We want to understand what is
really happening chemically when
an enzyme does its job.
 We’d also like to know how
biochemists probe these systems.

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Mechanism Topics
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Inhibitors,
concluded:
Pharmaceuticals
Mechanisms:
Terminology
Transition States
Enzyme chemistry
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Diffusion-controlled
Reactions
Binding Modes of
Catalysis
Induced-fit
Tight Binding of
Ionic Intermediates
Serine proteases
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Most pharmaceuticals are
enzyme inhibitors
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Some are inhibitors of enzymes that are
necessary for functioning of pathogens
Others are inhibitors of some protein
whose inappropriate expression in a
human causes a disease.
Others are targeted at enzymes that are
produced more energetically by tumors
than they are by normal tissues.
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Characteristics of
Pharmaceutical Inhibitors
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Usually competitive, i.e. they raise Km
without affecting Vmax
Some are mixed, i.e. Km up, Vmax down
Iterative design work will decrease Ki
from millimolar down to nanomolar
Sometimes design work is purely blind
HTS; other times, it’s structure-based
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Amprenavir
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Competitive inhibitor of HIV
protease,
Ki = 0.6 nM for HIV-1
No longer sold: mutual
interference with rifabutin,
which is an antibiotic used
against a common HIV
secondary bacterial infection,
Mycobacterium avium
10/23/2008
Qui ckT ime™ and a
T IFF (Uncompres sed) dec ompres sor
are needed to s ee this pic ture.
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When is a good inhibitor a
good drug?
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It needs to be bioavailable and nontoxic
Beautiful 20nM inhibitor is often neither
Modest sacrifices of Ki in improving
bioavailability and non-toxicity are okay if
Ki is low enough when you start
sacrificing
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How do we lessen toxicity
and improve bioavailability?
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Increase solubility…
that often increases Ki because the van
der Waals interactions diminish
Solubility makes it easier to get the
compound to travel through the
bloodstream
Toxicity is often associated with fat
storage, which is more likely with
insoluble compounds
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Drug-design timeline
2 years of research, 8 years of trials
-8
Research
0
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2
100
Cost/yr, 106 $
-3
Preliminary toxicity testing

10
Clinical Trials
Time, Yrs
Biochemistry: Enzyme Mechanisms
10
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Atomic-Level Mechanisms
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We want to understand atomic-level
events during an enzymatically
catalyzed reaction.
Sometimes we want to find a way to
inhibit an enzyme
in other cases we're looking for more
fundamental knowledge, viz. the ways
that biological organisms employ
chemistry and how enzymes make
that chemistry possible.
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How we study mechanisms

There are a variety of
experimental tools available for
understanding mechanisms,
including isotopic labeling of
substrates, structural methods,
and spectroscopic kinetic
techniques.
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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: scheme 6.1
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
(scheme 6.2)
Transition state has five ligands;
inherently less stable than scheme
6.1
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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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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
Intermediate electron acceptors and donors are
organic moieties or metals
Ultimate electron acceptor in aerobic organisms
is usually dioxygen (O2)
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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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Overcoming the barrier
Free Energy

Simple system:
single high-energy transition state
intermediate between reactants,
products
G‡
R
P
Reaction Coordinate
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Intermediates

Often there is a quasi-stable intermediate
state midway between reactants &
products; transition states on either side
Free Energy
T2
T1
I
R
P
Reaction Coordinate
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Activation energy &
temperature
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It’s intuitively sensible that higher
temperatures would make it easier
to overcome an activation barrier
Rate k(T) = Q0exp(-G‡/RT)
G‡ = activation energy or
Arrhenius energy
This provides tool for measuring
G‡
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Biochemistry: Enzyme Mechanisms
Svante
Arrhenius
p. 20 of 63
Determining G‡
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ln k
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Remember
k(T) = Q0exp(-G‡/RT)
ln k = lnQ0 - G‡/RT
Measure reaction rate
as function of
temperature
Plot ln k vs 1/T; slope
will be -G‡/R
1/T, K-1
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How enzymes alter G‡
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Enzymes reduce G‡ by
allowing the binding of the
transition state into the active
site
Binding of the transition state
needs to be tighter than the
binding of either the reactants
or the products.
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G‡ and Entropy
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Effect is partly entropic:
When a substrate binds,
it loses a lot of entropy.
Thus the entropic disadvantage of (say)
a bimolecular reaction is soaked up in
the process of binding the first of the
two substrates into the enzyme's active
site.
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Enthalpy and transition states
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Often an enthalpic component to
the reduction in G‡ as well
Ionic or hydrophobic interactions
between the enzyme's active site
residues and the components of
the transition state make that
transition state more stable.
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Two ways to change G‡
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Reactants bound by
enzyme are properly
positioned
Get into transitionstate geometry more
readily
E A
B
A+B
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Transition state is
stabilized
E A
B
A+B
A-B
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A-B
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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 guanadinium +1
Ser —CH2OH 0
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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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Cleavages by base
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Simple cleavage:

—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

HO
O
H
H :B
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H—B+
Biochemistry: Enzyme Mechanisms
: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+
+ OH-  R—OH  R—OH2+
(Slow)
(Fast)
 R+ + H2O
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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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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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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 of an enzyme. A diffusioncontrolled reaction will have a catalytic
efficiency on the order of 108 M-1s-1.
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Triosephosphate isomerase
(TIM)

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 or fig. 6.7.
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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
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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: Enzyme Mechanisms
William
Jencks
p. 37 of 63
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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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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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
10/23/2008
R1
NH
C
C
CH
NH
O
R-1Mechanisms
Biochemistry:
Enzyme
p. 45 of 63
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

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

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-
10/23/2008
Diagram courtesy of
Mariusz Jaskolski,
U. Poznan
Biochemistry: Enzyme Mechanisms
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Papain active site
Diagram courtesy
Martin Harrison,
Manchester University
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