Download - Chapter 6 Slides (Part I) CH490
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)'
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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)