11/02/2010biochem: enzyme mechanisms enzyme mechanisms andy howard introductory biochemistry 2...

11/02/2010Biochem: Enzyme Mechanisms

Enzyme mechanisms

Andy HowardIntroductory Biochemistry

2 November 2010

11/02/2010 Biochem: Enzyme Mechanisms P. 2 of 49

More about mechanisms

Many enzymatic mechanisms involve either covalent catalysis or acid-base interactions

We’ll give some examples of several mechanistic approaches


Mechanism Topics

Enzyme dynamics Enzyme chemistry Transition-state binding

Diffusion-controlled Reactions

Binding Modes of Catalysis

Redox reactions

Induced fit Ionic intermediates

Active-site amino acids

Serine proteases Reaction How they illustrate what we’ve learned

Specificity Evolution


The protein moves as well!

Changes to active-site conformation: 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


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


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.


Proline racemase

Pyrrole-2-carboyxlate resembles planar transition state


Yeast aldolase

Phosphoglycolohydroxamate binds much like the transition state to the catalytic Zn2+


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.


ADA transition-state analog

1,6 hydrate of purine ribonucleoside binds with KI ~ 3*10-13 M


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 (or the specificity constant) of an enzyme. A diffusion-controlled reaction will have a catalytic efficiency on the order of 108 M-1s-1.


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

Cartoon courtesy Wikibooks.org


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


Attack on Acyl Group

Transfer of an acyl group: section 14.6

Nucleophile Y attacks carbonyl carbon, forming tetrahedral intermediate

X- is leaving group


Direct Displacement 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


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


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


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)


Low-barrier H-bonds

Ordinary H-bonds buy us 10-30 kJ mol-1

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


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


Redox, continued

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


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


One-electron redox reactions

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


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


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


Hexokinase structure Diagram courtesy E. Marcotte, UT Austin


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


Reactive sidechains in a.a.’s

AA Group Charge@pH=7

Functions

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 guanidinium

+1 Anion binding

Ser —CH2OH 0 See cys


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


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


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


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


iClicker Quiz question 2What 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.


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


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 amino acids at position 1, 2, -1, . . .

Others are more promiscuous

NHCH

R1C

O

NH

CH

C

NH

R-1

O


Mechanism 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 near-neutral pH

Resulting + charge on His is stabilized by its proximity to a nearby carboxylate group on an aspartate side-chain.


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.


Diagram of first three steps


Diagram of last four steps

Diagrams courtesy University of Virginia


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


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 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.


Oxyanion hole cartoon

Cartoon courtesy Henry Jakubowski, College of St.Benedict / St.John’s University


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


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.


Chymotrypsin active site

Comfortably accommodates aromatics at S1 site

Differs from other mammalian serine proteases in specificity

Diagram courtesy School of Crystallography, Birkbeck College


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

Primary differences are in P1 side chain pocket, but that isn’t inevitable


Convergent evolution Reappearance of ser-his-asp triad in unrelated settings

Subtilisin: externals very different from mammalian serine proteases; triad same


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.


iClicker question #3Which 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.

11/02/2010biochem: enzyme mechanisms enzyme mechanisms andy howard introductory biochemistry 2...

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