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11/3/2009Biochem: Enzymes III
Enzymes III
Andy HowardIntroductory Biochemistry
3 November 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 Types of inhibitors
Kinetics of inhibition
Pharmaceuticals
What makes an inhibitor a useful drug?
Mechanisms:Terminology
Transition States
Stabilization of Transition States
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Distinctions we can make
Inhibitors can be reversible or irreversible
Where do they bind? At the enzyme’s active site At a site distant from the active site.
To what do they bind? To the unliganded enzyme E To the enzyme-intermediate complex or the enzyme-substrate complex (ES)
To both (E or ES)
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Types of inhibitors Irreversible Inhibitor binds without possibility of release
Usually covalent Each inhibition event effectively removes a molecule of enzyme from availability
Reversible Usually noncovalent (ionic or van der Waals)
Several kinds Classifications somewhat superseded by detailed structure-based knowledge of mechanisms, but not entirely
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Types of reversible inhibition
Competitive Inhibitor binds at active site of unliganded enzyme
Prevents binding of substrate Noncompetitive
Inhibitor binds distant from active site (E or ES)
Interferes with turnover Uncompetitive (rare?)
Inhibitor binds only to ES complex Removes ES, interferes with turnover
Mixed(usually Competitive + Noncompetitive)
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How to tell them apart Reversible vs irreversible
dialyze an enzyme-inhibitor complex against a buffer free of inhibitor
if turnover or binding still suffers, it’s irreversible
Competitive vs. other reversible: Structural studies if feasible Kinetics
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Competitive inhibition Put in a lot of
substrate:ability of the inhibitor to getin the way of the binding is hindered:out-competed by sheer #s of substrate molecules.
This kind of inhibition manifests itself as interference with binding, i.e. with an increase of Km
SIc
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Competitive inhibitors don’t affect turnover If the substrates manages to bind even though there is inhibitor present, then it can be turned over just as quickly as if the inhibitor is absent; so the inhibitor influences binding but not turnover.
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Kinetics of competition Competitive inhibitor hinders binding
of substrate but not reaction velocity:
Affects the Km of the enzyme, not Vmax.
Which way does it affect it? Km = amount of substrate that needs to be present to run the reaction velocity up to half its saturation velocity.
Competitive inhibitor requires us to shove more substrate into the reaction in order to achieve that half-maximal velocity.
So: competitive inhibitor increases Km
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L-B: competitive inhibitor
Km goes up so -1/ Km moves toward origin
Vmax unchanged so Y intercept unchanged
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Competitive inhibitor:Quantitation of Ki Define inhibition constant Ki to be the concentration of inhibitor that increases Km by a factor of two.
Km,obs = Km(1+[Ic]/Ki)
So [Ic] that moves Km halfway to the origin is Ki.
If Ki = 100 nM and [Ic] = 1 µM, then we’ll increase Km,obs elevenfold!
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Don’t get lazy!
A competitive inhibitor doesn’t automatically double Km
The amount by which the inhibitor increases Km is dependent on [I]c
If it happens that [I]c = KI, then Km will double, as the equation shows
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Noncompetitive inhibition Inhibitor binds distant from
active site, so it binds to theenzyme whether the substrateis present or absent.
Noncompetitive inhibitor has no influence on how available the binding site for substrate is, so it does not affect Km at all
However, it has a profound inhibitory influence on the speed of the reaction, i.e. turnover. So it reduces Vmax and has no influence on Km.
SI
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L-B for non-competitives
Decrease in Vmax 1/Vmax is larger X-intercept unaffected
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Ki for noncompetitives
Ki defined as concentration of inhibitor that cuts Vmax
in half Vmax,obs =Vmax/(1 + [In]/Ki)
In previous figure the “high” concentration of inhibitor is Ki
If Ki = Ki’, this is pure noncompetitive inhibition
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Uncompetitive inhibition
Inhibitor binds only if ES has already formed
It creates a ternary ESI complex This removes ES, so by LeChatlier’s Principle it actually drives the original reaction (E + S ES) to the right; so it decreases Km
But it interferes with turnover so Vmax goes down
If Km and Vmax decrease at the same rate, then it’s classical uncompetitive inhibition.
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L-B for uncompetitives -1/Km moves away from origin 1/Vmax moves away from the origin Slope ( Km/Vmax) is unchanged
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Ki for uncompetitives
Defined as inhibitor concentration that cuts Vmax or Km in half
Easiest to read from Vmax value Vmax,obs = Vmax/(1+[I]u/KI) Iu labeled “high” is Ki in this plot
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iClicker quiz, question 1
1. Treatment of enzyme E with compound Y doubles Km and leaves Vmax unchanged. Compound Y is: (a) an accelerator of the reaction
(b) a competitive inhibitor (c) a non-competitive inhibitor (d) an uncompetitive inhibitor
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iClicker quiz, question 2 2. Treatment of enzyme E with compound X doubles Vmax and leaves Km unchanged. Compound X is: (a) an accelerator of the reaction
(b) a competitive inhibitor (c) a non-competitive inhibitor (d) an uncompetitive inhibitor
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Mixed inhibition
Usually involves interference with both binding and catalysis
Km goes up, Vmax goes down Easy to imagine the mechanism:
Binding of inhibitor alters the active-site configuration to interfere with binding, but it also alters turnover
Same picture as with pure noncompetitive inhibition, but with Ki ≠ Ki’
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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
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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
Cost/yr, 106 $
10
100
Improving affinity
Toxicity and
bioavailabili
ty
Research Clinical Trials
Preliminary toxicity testing
Stage I clinical trials
Stage II clinical trials
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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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Overcoming the barrier Simple system:single high-energy transition state intermediate between reactants, products
Free Energy
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
Free Energy
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.
In fact, the enzyme must stabilize the transition-state complex EX‡ more than it stabilizes the substrate complex ES (see section 14.2).
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Dissociation constants for ES and EX* Dissociation constant for ES:Ks = [E][S]/[ES]
Dissociation constant for EX‡:KT = [E][X‡]/[EX‡]
Transition state theory says the ratio of reaction rates is related to the ratio of these:
ke/ku = Ks / KT
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What makes EX‡ more stable than ES? Intrinsic (enthalpic) binding energy
of ES makes it a lower-energy species than E+S; but we want EX* to be lower.
ES loses entropy relative to E + S ES is sometimes strained, distorted, or desolvated relative to E+S
So if EX‡ is less strained and has more entropy, we win
See section 14.3
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How tight is the binding?
Section 14.4 gives some examples Transition-state analogs are stable molecules that are geometrically and electrostatically similar to transition states
Sometimes the analogs bind ~ 160 - 40000 times more avidly than substrates
1,6-hydrate of purine nucleoside binds to adenosine deaminase with KI = 3*10-
13M
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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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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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Near-Attack Conformations
Substrate is preorganized in the active site such that the reacting atoms are in van der Waals contact and at an angle resembling the bond to be formed in the transition state.
The NAC would form anyway (0.0001% of the time?) but with the help of the enzyme, it forms 1-70% of the time