CHAPTER 6 Enzymes

Two$Fundamental$Condi0ons$of$Life,$

•  Ability'to'self,replicate'o  (Covered'in'Spring'Quarter)'

•  Ability'to'catalyze'chemical'reac>ons'selec>vely'and'effec>vely'o  (Covered'here'and'extensively'in'Winter'Quarter)'

$

Enzymes

Consider)Sugar…) •  Conversion'to'Energy'=',ΔG…'But'slow!'•  Requires'catalysis'in'all'living'beings''

Enzymes

What are they & why do we care!

Enzymes – What are they & why do we care!

Enzymes)are)cataly6cally)ac6ve)biological)macromolecules)• Many'enzymes'are'globular'proteins,'

o  But'there'are'ribozymes'(RNA;'protein/RNA'hybrids)'o  Some'are'membrane,associated'

• Enzymology'is'the'oldest'field'of'biochemistry'o  da>ng'back'to'late'1700s'

Enzymes)are)fundamental)to)all)biological)processes)•  Control'metabolism'and'catabolism'•  Form'macromolecules'from'small'precursors''•  As'we'just'saw,'provide'energy'for'movement'(Ac>n/Mysosin)'•  etc..'

Enzymes – What are they & why do we care!

Enzymes)are)important)for)understanding)disease)•  Muta>on'leads'to'poor'func>on,'or'enhanced'func>on'

•  Many'drugs'target'enzymes,'because'of'precise'control'over'physiological'func>on'

Enzymes)are)desirable)for)driving)chemical)reac6ons)•  Degree'of'efficiency'and'specificity'is'o[en'far'greater'than'

anything'achieved'in'synthe>c'chemistry.'And'do'so'under'very'mild'condi>ons'(neutral'pH'and'in'water)''

•  Applica>ons'in'the'fields'of'biotechnology,'bioengineering,'green'chemistry'&/or'synthe>c'biology'

Enzymes – What are they & why do we care!

Here)we)will)give)an)introduc6on)to)the)fundamentals)regarding)

structure)func6on)rela6onship)to)enzyme)mechanism)

'o  Next)Quarter)–'will'focus'heavily'on'enzyme'kine>cs'&'

enzyma>c'pathways'

Enzymes – What are they & why do we care!

1897)–)Eduard)Buchner)discovered)that)yeast)

extracts)could)ferment)sugar)to)alcohol)))

•  First'demonstra>on'that'molecules'separated'from'the'organism'could'catalyze'a'chemical'reac>on'

o  Previously'thought'to'be'a'inseparable'property'of'the'organism'

•  Alcohol'dehydrogenase'from'yeast'was'one'of'the'first'enzymes'purified'(1937)'

o  Homolog'from'horse'was'one'of'the'first'3D'structures'determined'(1973)'

Image:'www.rcsb.org'

Yeast$ADH$

Enzyme Classification

All)Enzymes)are)‘Catalysts’)'Increase(the(rate(of(reac-on(by(lowering(the(ac-va-on(energy((ΔG‡)((

Organized(into(six)classes)–(by(type(of(chemistry(they(catalyze(

Enzymes: Why biocatalysts over inorganic catalysts?

In(a(synthe-c(chemistry(lab(we(o?en(u-lize(inorganic(catalysts…(((((So(why(does(nature(use(enzymes?(•  Greater$reac0on$specificity:'avoids'side'products'•  Milder$reac0on$condi0ons:'conducive'to'condi>ons'in'cells'•  Higher$reac0on$rates:'in'a'biologically'useful'>meframe'•  Capacity$for$regula0on:'control'of'biological'pathways'

COO

OHO COO

COO

O COO

NH2

OOCCOO

O

OH

OH

COO

NH2

COO

-

-

-

-

-

-

--

Chorismate mutase

•  Metabolites$have$many$poten0al$pathways$of$decomposi0on$$

$•  Enzymes$make$the$desired$one$most$favorable$

We'come'back'to'this'one'later…'

Enzymes

How do they work?

How Do Enzymes Work: Substrate specificity

Enzyme)chemistry)takes)place)within)a)binding)pocket)•  Highly)controlled)environment)

o  Electrosta-cs(o  Hydrophobic(o  Hydrated/Anhydrous(o  Steric(Interac-ons(o  Stereochemistry(o  Etc…(

Ac0ve$Site$Pocket$

Enzyme)chemistry)takes)place)within)a)binding)pocket)•  Highly)controlled)environment)

Example:(Phenylalanine(hydroxylase(

OOC NH3

H

OOC NH3

H

H NH

HOH

OH

H

OH

CH3

OOC NH3

HOH

--

-

+

+

+

No)binding)

OOC NH3

H

OOC NH3

H

H NH

HOH

OH

H

OH

CH3

OOC NH3

HOH

--

-

+

+

+

)))Binding)but)no)reac6on)

OOC NH3

H

OOC NH3

H

H NH

HOH

OH

H

OH

CH3

OOC NH3

HOH

--

-

+

+

+

How Do Enzymes Work: Substrate specificity

How Do Enzymes Work: Enhanced Reaction Rates

Remember)Sugar…)

Many(biological(reac-ons(are(energe-cally(favorable,(but(kine-cally(very(slow…(

How Do Enzymes Work: Enhanced Reaction Rates

Remember)Sugar…)

Enzymes((like(all(catalysts)(increase(the(rate(of(reac-on(by(lowering(the(transi-on(state.((Capable(of(rate(enhancements(on(the(order(or(105(–(1017(x(faster(than(uncatalyzed(reac-ons((We’ve(seen(this(before,(but(let’s(look(more(closely(at(how(this(happens…(

How to Lower ΔG‡: Entropic Effects

Enzymes)organize)reac6ve)groups)into)close)

proximity)and)proper)orienta6on)

•  Uncatalyzed$bimolecular$reac0ons$$','two'free'reactants'→'single'restricted'transi>on'state''','conversion'is'entropically'unfavorable'

'

• $Uncatalyzed$unimolecular$reac0ons$$','flexible'reactant'→'rigid'transi>on'state'','conversion'is'entropically'unfavorable'

'

• $Catalyzed$reac0ons$','Enzyme'uses'the'binding'energy'of'substrates'to'organize'the'reactants'to'a''''fairly'rigid'Enzyme,Substrate'(ES)'complex'','Entropy'cost'is'paid'during'binding'','Rigid'ES'complex'→'transi>on'state'conversion'is'entropically'OK'

How to Lower ΔG‡: The Proximity Model

Support)for)the)Proximity)Model)

Rate'enhancement'is'a'product'of'entropy'reduc>on''

Example:)Anhydride'forma>on'from'esters'and'carboxylates'

Rate(enhancement(shows(strong(dependence(on(proximity(of(the(two(reac-ve(groups(

Largely$ΔS$effect$

How to Lower ΔG‡: Transitions State Stabilization

Enzymes)bind)transi6on)states)best)

•  The'idea'was'proposed'by'Linus'Pauling'in'1946'o  Enzyme'ac>ve'sites'are'complimentary'to'the'transi>on'state'of'the'reac>on'

o  Enzymes'bind'transi>on'states'beker'than'substrates'o  Interac>ons'with'the'transi>on'state'as'compared'to'the'ground'state'lower'the'ac>va>on'barrier'

Largely)ΔH)effect)

How to Lower ΔG‡: Transitions State Stabilization

Consider$magnets$=$electrosta0c$aa’s$

How to Lower ΔG‡: Transitions State Stabilization

Support)for)the)TS)Stabiliza6on)Model)'

Example:)Ester'hydrolysis'

Stable)structural)analogs)of)transi6on)

states)bind)more)strongly)than)reactants)

)

Good)tools)for)studying)cataly6c)

mechanism)&)for)drug)design!)

TS)Analog)

How to Lower ΔG‡: Combined ΔS & ΔH Effects

Chorismate(mutase(

Unfavorable'conforma>on' TS'state'Substrate' Product'

Burschowsky)et)al.)PNAS)2014)'

3'Crystal'Structures'of'Chorismate'mutase'•  Substrate'Bound'•  Product'Bound'•  Transi>on'State'Analog'Bound''

Enzymes)manipulate)both)ΔS)and)ΔH)

How to Lower ΔG‡: Combined ΔS & ΔH Effects

Enzymes)manipulate)both)ΔS)and)ΔH)

Chorismate(mutase(

Unfavorable'conforma>on' TS'state'Substrate' Product'

Arg/$

Binding)pocket)reduces)

entropy)of)substrate)

)

Electrosta6c)complementarity)

stabilizes)transi6on)state)

Enzymes

Catalytic Mechanisms

Catalytic Mechanisms

o  acid,base'catalysis:'give'and'take'protons'o  covalent'catalysis:'change'reac>on'paths'o  metal'ion'catalysis:'use'redox'cofactors'

Acid-Base Catalysis: Chemical Example

Consider)ester)hydrolysis:)

Enzymes can do acid and base catalysis simultaneously •  Reaction takes place within a binding pocket •  Utilize both acidic and basic A.A.’s

R

O

C

H

3

O

R

O

C

H

3

O

O

H

H

+

R

O

O

H

+ H-OH + + CH3OH

•  Water'is'a'poor'nucleophile,'and'methanol'is'a'poor'leaving'group'

•  Aqueous'hydrolysis'can'be'catalyzed'either'by'acids'or'by'bases'

Acid-Base Catalysis

Example:)amide'hydrolysis'

Exothermic$Reac0on$(~2$kCal/mol)$•  Resonance'makes'amide'bond'

kine>cally'stable'

Specific Acid-Base Catalysis: Water Catalysis

Can)be)catalyzed)with)OH[)or)H3O+)

)

Within)an)enzyme)binding)pocket)water)

may)be)removed)

Example:)amide'hydrolysis'

General Acid-Base Catalysis: Non-water catalysis

Example:)amide'hydrolysis'

In)the)Enzyme)Binding)Pocket)

Both)Acid)and)Base)Contribute)to)Catalysis)

General Acid-Base Catalysis

Some)of)the)Major)Players)in)General)Acid[Base)Catalysis)

Note:)Cys,(Ser,(Tyr(are(included(because(pKa’s(can(be(greatly(shi?ed(within(a(cataly-c(binding(pocket!(

Covalent Catalysis

A)transient)covalent)bond)between)the)enzyme)and)the)substrate)are)used)to)change)the)reac6on)pathway)

)

•  Reac>on'Pathways''

o  Uncatalyzed:''''

o  Catalyzed:'

•  Requires'a'nucleophile'on'the'enzyme'o  Can'be'a'reac>ve'serine,'thiolate,'amine,'or'carboxylate'from'amino'acid'or'co,factor'

o  Nucleophile'is'always'regenerated'a[er'reac>on'is'complete'

A–B'',,!''A''+''B'H2O'

intermediate)

A–B''+''X:'',,!''A–X''+''B' ,,!'A''+''X:'+''B'H2O'

Metal Ion Catalysis

Involves)a)metal)ion)bound)to)the)enzyme)'

•  Interacts'with'substrate'to'facilitate'binding'o  Stabilizes'nega>ve'charges'

•  Par>cipates'in'oxida>on'reac>ons'

~1/3)of)all)enzymes)

u6lize)metals!)

Catalytic Mechanisms

o  acid,base'catalysis:'give'and'take'protons'o  covalent'catalysis:'change'reac>on'paths'o  metal'ion'catalysis:'use'redox'cofactors'

Most)enzymes)u6lize)a)combina6on)of)these)mechanisms)to)carry)out)catalysis)