10/23/2008 biochemistry: enzyme mechanisms 1 enzyme mechanisms andy howard introductory...
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10/23/2008Biochemistry: Enzyme Mechanisms 1
Enzyme Mechanisms
Andy HowardIntroductory Biochemistry, Fall 2008
Thursday 23 October 2008
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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
Inhibitors, concluded:Pharmaceuticals
Mechanisms:Terminology
Transition States Enzyme chemistry
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
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
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
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
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
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When is a good inhibitor a good drug? 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?
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
log
Ki
Time, Yrs 1020
-3
-8
Co
st/y
r, 1
06 $
10
100
Imp
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To
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ity
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av
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bili
ty
Research Clinical Trials
Pre
limin
ary
to
xic
ity
te
stin
g
Sta
ge
I clin
ical
tri
als
Sta
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10/23/2008 Biochemistry: Enzyme Mechanisms p. 10 of 63
Atomic-Level Mechanisms 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 Define them as reactions that involve
charged, or at least polar, intermediates Typically 2 reactants
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 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 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 Both electrons stay with one atom
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 Both end up as radicals R1O—OR2 R1O• + •OR2
Radicals are highly reactive—some more than others
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Oxidation-Reduction Reactions
Commonplace in biochemistry: EC 1 Oxidation is a loss of electrons Reduction is the gain of electrons In practice, often:
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 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 Simple system:
single high-energy transition state intermediate between reactants, products
Fre
e E
ner
gy
Reaction Coordinate
RP
G‡
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Intermediates Often there is a quasi-stable intermediate
state midway between reactants & products; transition states on either side
Fre
e E
ner
gy
R P
T1T2
I
Reaction Coordinate
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Activation energy & temperature
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‡
Svante Arrhenius
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Determining G‡
Rememberk(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
ln k
1/T, K-1
uncatalyzed
catalyzed
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How enzymes alter G‡
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
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 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‡
Reactants bound by enzyme are properly positioned
Get into transition-state geometry more readily
Transition state is stabilized
E AB
E AB
A+B A+BA-B A-B
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Reactive sidechains in a.a.’s AA Group Charge
@pH=7Functions
Asp —COO- -1 Cation binding, H+ transfer
Glu —COO- -1 Same as above
His Imidazole ~0 Proton transfer
Cys —CH2SH ~0 Covalent binding of acyl gps
Tyr Phenol 0 H-bonding to ligands
Lys NH3+ +1 Anion binding, H+ transfer
Arg guanadinium +1 Anion binding
Ser —CH2OH 0 See cys
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Generalizations about active-site amino acids Typical enzyme has 2-6 key catalytic
residues His, asp, arg, glu, lys account for 64% Remember:
pKa values in proteins sometimes different from those of isolated aa’s
Frequency overall Frequency in catalysis
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Cleavages by base 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:
—C—N
O
—C—N
O-
HO
H
:
:
:B
HOH—B+
—C—OH
O
+ HN
:B
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Cleavage by acid
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+
R+ + H2O
(Slow)
(Fast)
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Covalent catalysis 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 Net reaction:
Sucrose + Pi Glucose 1-P + fructose Fructose=A, Glucose=X, Phosphate=B
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Rates often depend on pH 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
Papain pH-rate profile
0
1
2 3 4 5 6 7 8 9 10 11
pH
relative reaction rate
Cys-25 His-159
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Diffusion-controlled reactions 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 diffusion-controlled 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
Km=10µMkcat=4000s-1kcat/Km=4*108M-1s-1
DHAP
Glyc-3-P
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TIM mechanism 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
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.
William Jencks
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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.
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Adenosine deaminase with transition-state analog Transition-state analog:
Ki~10-8 * substrate Km
Wilson et al (1991) Science 252: 1278
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
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Induced fit Refinement on original Emil
Fischer lock-and-key notion: both the substrate (or transition-
state) and the enzyme have flexibility
Binding induces conformational changes
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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
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Hexokinase structure Diagram courtesy E. Marcotte, UT Austin
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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
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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
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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
NH
CH
R1C
O
NH
CH
C
NH
R-1
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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 near-neutral 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 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 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 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 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 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 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
10/23/2008 Biochemistry: Enzyme Mechanisms p. 57 of 63
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
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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
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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
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Convergent evolution 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 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 Ancestrally related to ser
proteases? Cathepsins, caspases,
papain Contrasts:
Cys —SH is more basicthan ser —OH
Residue is less hydrophilic S- is a weaker nucleophile
than O-
Diagram courtesy ofMariusz Jaskolski,U. Poznan
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Papain active site
Diagram courtesy Martin Harrison,Manchester University