10/23/2008 biochemistry: enzyme mechanisms 1 enzyme mechanisms andy howard introductory...

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

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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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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

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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