Proximity Effects on Reaction Ratessites.fas.harvard.edu/~lsci1a/11-14.pdf · 2 Orientation Effects...
Transcript of Proximity Effects on Reaction Ratessites.fas.harvard.edu/~lsci1a/11-14.pdf · 2 Orientation Effects...
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Lectures 15-16: The Molecular Basis of Enzyme Catalysis: HIV Protease
1. The function and structure of HIV proteasea. Introduction to proteasesb. Discovery of HIV proteasec. Overview of the three-dimensional structure of HIV protease
2. Chemical reactions and the energies driving thema. Amide bond cleavage: the reaction catalyzed by HIV proteaseb. Thermodynamics of a reaction and free energyc. Kinetics of a reaction and ΔG‡
d. Reaction energy diagramse. Transition states, intermediates, and how to draw them
3. How enzymes accelerate chemical reactions: the case of HIV proteasea. Catalysts alter a reaction’s kinetics, but not its thermodynamicsb. Chemical strategies behind enzyme catalysis
i. Proximity and orientation effectsii. Nucleophilicity and electrophilicityiii. Acid and base catalysis
4. The molecular basis of substrate specificitya. Trypsin substrate specificityb. HIV protease substrate specificity
O
O
OO
O
N
O
O
A
Proximity Effects on Reaction Rates
Rate at 1 M concentrations = 4 x 10-6 M/s
O
OO
N
O
O
B A B C
OOO
O
N
O
O
Rate = 0.8 M/s (200,000-fold faster!)
• Intramolecular reactions are much faster than intermolecular ones
A B
O
O
O
O
N
O
O
CA B
2
Orientation Effects
A B
A B
A B
Increasing rateof reaction
• The fewer nonproductive ways two groups can be oriented,the faster they will react
A
B
A
B
A
B
(assume A and B reactupon sideways collision)
Proximity and Orientation Effectsin HIV Protease
O
NH
R1HN
R2O O
HN
O
O
HN
R2O
HOH
NH
R1
O
HNH
+ H+
protein substrate
“attacking” water
HIV proteasebackbone
(Ile 50 and Ile 50')
• The enzyme’s activesite holds substrates(protein & water) inclose proximity
O C
N H
• HIV protease useshydrogen bonds toorient substratesproductively
H
O
H
N
O
HN
O
H
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A Network of InteractionsPrecisely Positions the
Substrates of HIV Protease
Note: structure isof an inactiveAsn 25 mutant ofHIV proteasecomplexed witha substrate.
Nucleophiles and Electrophiles
Nu–E+Electrophile
electron-deficient,“likes electrons”
NE
Nucleophileelectron-rich,“likes nuclei”
• Nucleophilicity and electrophilicity are kinetic parameters• The more nucleophilic the nucleophile, or electrophilic the
electrophile, the faster the reaction (by definition!)
bond-formingreaction
u
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Factors Governing Nucleophilicity
1) More basic molecules tend to be more nucleophilicwhen the nucleophilic atoms are of comparable size:
HO
H
Poor nucleophilepKa of conjugate acid = –1.5
Good nucleophilepKa of conjugate acid = 15.5
OH
2) Larger atoms (those lower on the periodic table) makebetter nucleophiles:
Weaker nucleophile Stronger nucleophileHS
HHO
H
Electrophiles in the Molecules of Life
Peptide hydrolysis
• The most common electrophiles in the chemistry of life areC=O and P=O
• Groups that are more electron-poor are more electrophilicand therefore react more quickly with nucleophiles
O
OO
P
O
O
O
O
NH
Translation DNA polymerizationDNA hydrolysis
Protein phosphorylation
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Acids and Bases Can EnhanceElectrophilicity and Nucleophilicity
HO
HO
H
Better nucleophile
more electron-richBASE
:OH-
O
HN
Better electrophile
more electron-poor
ACID
H+
O
HN
H
O
HN
H
OH O
HN
O H
H
Base Catalysis of Amide Hydrolysis
Weaker nucleophile:slower reaction,
higher ΔG‡
‡
!+!"
O
HN
H
O
‡O
HN
O H!"H2O + base
Better nucleophile:faster reaction,
lower ΔG‡
δ−
• Base can accelerate amide hydrolysis by deprotonatingwater, increasing its nucleophilicity
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O
HN
O
O
H
O
OH
O HO
HN
O
O
H
O
O
O H
H
O
HN
O
O
H
O
OH
OH
Base Catalysis by HIV Protease
‡
Asp 25of HIV protease
!" δ−
Asp 25of HIV protease
• HIV protease precisely positions Asp 25 to serve as a baseto deprotonate water
• The enhanced nucleophilicity of deprotonated wateraccelerates amide hydrolysis
deprotonationof water
O
HN
O H
H
O
HN
H
OH
Acid Catalysis of Amide Hydrolysis‡
!"δ+
Weaker electrophile:slower reaction,
higher ΔG‡
O
HN
H
OHH
O
HN
O H
H
H
‡
δ+
Stronger electrophile:faster reaction,
lower ΔG‡
• Acid can accelerate amide hydrolysis by protonating theamide oxygen, increasing its electrophilicity
δ+
amide+
acid
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O
HN
O
O
O
OH
O HHO
HN
O
O
H
O
O
O H
H
Acid Catalysis by HIV Protease
‡O
HN
O
O
H
O
OH
O H
Asp 25’of HIV
protease
!"δ−
Asp 25’of HIV
protease
• HIV protease precisely positions Asp 25’ to serve as anacid to protonate the substrate amide, increasing itselectrophilicity and accelerating amide hydrolysis
protonationof amide
Enzymes Can Catalyze Reactions in WaysThat Simple Acids and Bases Cannot
O
HN
H
OH
ACIDH +
BASE
:OH-
How can an acid and basesimultaneously catalyze a
single reaction? enzyme acid
O
HN
O
O
H
O
OH
OH
enzyme base
• Enzymes can simultaneously use acidic and basic groupsthat, in a flask, would wander and neutralize each other
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HIV Protease Active Site in Action
Asp 25’
Ile 50’ attackingwater
substrate
Note: locations of hydrogen atoms are usually inferred (and often not shown)
H
Asp 25
O
HN
O
O
H
O
OH
O H
HIV Protease Catalysis Summary
O
HN
O
O
H
O
OH
OH
‡O N
O
O
H
O
OH
O
H
H
O
O
O
OH
O
HN
O HH
Acidcatalysis
Basecatalysis
O
O
O
O
H
O
O
H
N
H
H
δ−δ−
O
HN
O
O
O
OH
O HH
‡
Acidcatalysis
Basecatalysis
δ−δ−
Tetrahedralintermediate
Asp 25’ Asp 25
Proximity andorientation effects
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Lectures 15-16: The Molecular Basis of Enzyme Catalysis: HIV Protease
1. The function and structure of HIV proteasea. Introduction to proteasesb. Discovery of HIV proteasec. Overview of the three-dimensional structure of HIV protease
2. Chemical reactions and the energies driving thema. Amide bond cleavage: the reaction catalyzed by HIV proteaseb. Thermodynamics of a reaction and free energyc. Kinetics of a reaction and ΔG‡
d. Reaction energy diagramse. Transition states, intermediates, and how to draw them
3. How enzymes accelerate chemical reactions: the case of HIV proteasea. Catalysts alter a reaction’s kinetics, but not its thermodynamicsb. Chemical strategies behind enzyme catalysis
i. Proximity and orientation effectsii. Nucleophilicity and electrophilicityiii. Acid and base catalysis
4. The molecular basis of substrate specificitya. Trypsin substrate specificityb. HIV protease substrate specificity
O
NH
P3HN
P4O O
HN
O
NH
P1
P2 O
HN
O
NH
P2'
P1' O
HN
O
NH
P4'
P3' O
HN
Protease Substrate Specificity
To C-terminal endof substrate
To N-terminal endof substrate
Scissilebond
S3 S1
S4 S2
S2’ S4’
S3’S1’
Enzyme specificitypockets recognize the
specific amino acidresidues surrounding the
bond to be hydrolyzed
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Trypsin: A Digestive Protease that CleavesSubstrates Containing Lys or Arg
trypsin activesite residues
enzymeAsp189
S1
scissile bond
substrateLys P1
substrate peptide
trypsinenzyme
+–
Basis of Trypsin Substrate Specificity
Asp 189
O O
NH2H2N
NH
NH
O
HN
O O
NH
O
HN
H3N
LysP1
ArgP1
Asp 189S1 S1
• The presence of anionic Asp 189 in the S1 site causes astrong preference for P1 to be a cationic Lys or Arg
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HIV Protease-Substrate Interactions
Animation rendered by Brian Tse
The HIV Protease ActiveSite: A Closer Look
Note: structure isof an inactiveAsn 25 mutant ofHIV proteasecomplexed witha substrate.
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HIV Protease Substrate Selectivity
• HIV protease recognizes more substrate amino acids thantrypsin, but without a strong preference at any one position
The 10 sites cleaved by HIV protease:
Key: MA = matrix; CA = capsid; NC = nucleocapsid; TF = trans frame peptide; PR = protease;AutoP = autoproteolysis (self-cleaving) site; RT = reverse transcriptase; RH = RNAse H; integrase = IN
O
NH
HN
O O
NO
NH
O
HN
HN
N NO
NH
HN
O
NH
H
HOH
H
H
H
H
HIV Protease Specificity: P1 and P3
Asn 25’
• HIV protease’sstructure can explainsome aspects of itssubstrate specificity
• P3 = polar orcharged; S3 containsa bound water
Arg P3
waterS3
Gly 49S1
Leu P1
• P1= large andhydrophobic;complementsS1 = Gly 49
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Key Points: HIV Protease & Enzyme Catalysis• HIV protease catalyzes polyprotein amide bond hydrolysis• Thermodynamics reflect the difference in energy between
reactants and products, as measured by ΔG°rxn
• Kinetics reflect reaction rates, determined by ΔG‡
• Enzymes lower ΔG‡ by using a variety of chemical strategiesto create a precise transition state-stabilizing active siteenvironment
• Enzymes use proximity and orientation effects to increasethe concentration of substrates, increasing rates of reactions
• Enzymes use acid and base catalysis to enhance thenucleophilicity or electrophilicity of reactants
• Specificity arises from protein-substrate interactions
Life Sciences 1aLecture Slides Set 10Fall 2006-2007Prof. David R. Liu
N N
HO OH
O
O
NH
O
HN
N
S S
N
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Lectures 17-18: The molecular basis of drug-protein binding:
HIV protease inhibitors
1. Drug development and its impact on HIV-infected patients
2. Energetic dissection of a small molecule binding to a protein
a. Enthalpy changes upon binding
b. Entropy changes upon binding
3. Case studies of saquinavir and ritonavir, two small-molecule HIV
protease inhibitors
a. Fill hydrophobic pockets with hydrophobic groups
b. Provide complementary hydrogen bond donors and acceptors
c. Mimic the transition state of a reaction
d. Maximize the rigidity of the drug
e. Displace bound water molecules Required: Lecture NotesMcMurray p. 808-810, 640-642
Lecture Readings
Impact of Anti-HIV Drugs
• 1990s: anti-HIV drugs transform HIV infection from a shortdeath sentence to a chronic (but very serious) illness
• 13 FDA-approved drugs inhibit HIV reverse transcriptase; 9drugs inhibit HIV protease (first approved December, 1995)
• Mortality rate of U.S. patients with advanced AIDS:• 29% per year in 1995• 9% per year in mid-1997
• 1997-2003: Death rate from AIDS in Europe falls 80%• Gains primarily attributed to combination therapy involving
HIV protease inhibitors + other antiretroviral agents
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Drug Development is Very Difficult
• Total cost to develop a drug = ~$1 billion + ~10-15 years
1) Potency (affinity)
Successful Drugs Must Satisfy ManyChemical and Biological Requirements
+Keq = Ka = 1÷Kd
drug-protein complexdrug protein target
targetnon-target non-target non-target
2) Specificity (toxicity, immunogenicity)
oralcellular
3) Bioavailability
k inactive or toxic4) Biostability 5) Economics