CHAPTER 6

Enzymes

Enzymes
Two$Fundamental$Condi0ons$of$Life,$
Ability'to'self,replicate'
o (Covered'in'Spring'Quarter)'

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

$
Consider)Sugar)

Conversion'to'Energy'=',G'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'eld'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'eciency'and'specicity'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'elds'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'
Yeast$ADH$

Alcohol'dehydrogenase'from'yeast'was'one'of'the'rst'
enzymes'puried'(1937)'
o Homolog'from'horse'was'one'of'the'rst'3D'
structures'determined'(1973)'

Image:'www.rcsb.org'

Enzyme Classification
All)Enzymes)are)Catalysts)
'

Increase(the(rate(of(reac-on(by(
lowering(the(ac-va-on(energy((G)(
(

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$reac0on$specicity:'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

OH

COO

NH2

We'come'back'to'
this'one'later'
COO

NH2
O

O
OH

COO

COO

Chorismate
mutase

OOC

OH

Metabolites$have$many$
COO
poten0al$pathways$of$
decomposi0on$$
$
COO Enzymes$make$the$
desired$one$most$
O
favorable$
-

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$

How Do Enzymes Work: Substrate specificity

Enzyme)chemistry)takes)place)within)a)binding)pocket)

Highly)controlled)environment)

Example:(Phenylalanine(hydroxylase(
H

H
H
+
OOC
+
NH
3 NH
OOC
+
OOC
3
NH3

H
+
OOC
NH3 +
+
NH3
OOC OOC
NH
-

H
H
H
+
NH3 +
- OOC
+
OOC
NH3
OOC
NH3

OH
OH
OH

No)binding)

OH
OH
OH
HO
OH
HO
OH
HH
O
OH
H
H
H
H
H H
H
NH
H
NH
NH
CH3
CH3
CH
3

)))Binding)but)no)reac6on)

How Do Enzymes Work: Enhanced Reaction Rates

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

Remember)Sugar)

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(
(
Weve(seen(this(before,(but(
lets(look(more(closely(at(how(
this(happens(

How to Lower G: 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$$
'

','exible'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 G: 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$S$eect$

How to Lower G: 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)H)eect)

How to Lower G: Transitions State Stabilization

Consider$magnets$=$electrosta0c$aas$

How to Lower G: 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 G: Combined S & H Effects

Enzymes)manipulate)both)S)and)H)
Chorismate(mutase(
Substrate'

Unfavorable'
conforma>on'

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

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

TS'state'

Product'

How to Lower G: Combined S & H Effects

Enzymes)manipulate)both)S)and)H)
Chorismate(mutase(
Substrate'

Binding)pocket)reduces)
entropy)of)substrate)
)
Electrosta6c)complementarity)
stabilizes)transi6on)state)

Arg/$

Unfavorable'
conforma>on'

TS'state'

Product'

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:)
O

O
R
O CH3

H-OH

OH
O

+ H

O
R

CH3OH

OH

CH3

Water'is'a'poor'nucleophile,'and'methanol'is'a'poor'
leaving'group'
Aqueous'hydrolysis'can'be'catalyzed'either'by'acids'or'by'
bases'

Enzymes can do acid and base catalysis simultaneously

Reaction takes place within a binding pocket
Utilize both acidic and basic A.A.s

Acid-Base Catalysis
Example:)amide'hydrolysis'

Exothermic$Reac0on$(~2$kCal/mol)$
Resonance'makes'amide'bond'
kine>cally'stable'

Specific Acid-Base Catalysis: Water Catalysis

Example:)amide'hydrolysis'

Can)be)catalyzed)with)OH[)or)H3O+)
)
Within)an)enzyme)binding)pocket)water)
may)be)removed)

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(pKas(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'
'

H2O'

o Uncatalyzed:'''' AB'',,!''A''+''B'
o Catalyzed:'

H2O'

AB''+''X:'',,!''AX''+''B' ,,!'A''+''X:'+''B'
intermediate)

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'

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)