Download Proximity Effects on Reaction Rates

Lectures 15-16: The Molecular Basis of Enzyme Catalysis: HIV Protease
1.
2.
3.
4.
The function and structure of HIV protease
a.
Introduction to proteases
b.
Discovery of HIV protease
c.
Overview of the three-dimensional structure of HIV protease
Chemical reactions and the energies driving them
a.
Amide bond cleavage: the reaction catalyzed by HIV protease
b.
Thermodynamics of a reaction and free energy
c.
Kinetics of a reaction and ΔG‡
d.
Reaction energy diagrams
e.
Transition states, intermediates, and how to draw them
How enzymes accelerate chemical reactions: the case of HIV protease
a.
Catalysts alter a reaction’s kinetics, but not its thermodynamics
b.
Chemical strategies behind enzyme catalysis
i.
Proximity and orientation effects
ii.
Nucleophilicity and electrophilicity
iii.
Acid and base catalysis
The molecular basis of substrate specificity
a.
Trypsin substrate specificity
b.
HIV protease substrate specificity
Proximity Effects on Reaction Rates
O
N O
O
O
O
O
O
A
O
N O
O
O
B
A
O
C
B
Rate at 1 M concentrations = 4 x 10-6 M/s
O
O
N
O
O
O
O
N O
O
O
O
O
O
A
B
A
B
C
Rate = 0.8 M/s (200,000-fold faster!)
• Intramolecular reactions are much faster than intermolecular ones
1
Orientation Effects
B
B
A
A
A
B
A
B
A
B
B
A
(assume A and B react
upon sideways collision)
Increasing rate
of reaction
• The fewer nonproductive ways two groups can be oriented,
the faster they will react
Proximity and Orientation Effects
in HIV Protease
O
H
N
O
R2
R1
N
H
H
N
+
H
O
H
O
O
O
N
H
N
H
H
R1
O
R2
H
+
H
N
H
H
N
O
O
O
• HIV protease uses
hydrogen bonds to
orient substrates
productively
O
H
N
O
HIV protease
backbone
(Ile 50 and Ile 50')
H “attacking” water
C
N H
protein substrate
• The enzyme’s active
site holds substrates
(protein & water) in
close proximity
2
A Network of Interactions
Precisely Positions the
Substrates of HIV Protease
Note: structure is
of an inactive
Asn 25 mutant of
HIV protease
complexed with
a substrate.
Nucleophiles and Electrophiles
Electrophile
electron-deficient,
“likes electrons”
E
+
–
Nu
Nucleophile
electron-rich,
“likes nuclei”
bond-forming
reaction
E
Nu
• Nucleophilicity and electrophilicity are kinetic parameters
• The more nucleophilic the nucleophile, or electrophilic the
electrophile, the faster the reaction (by definition!)
3
Factors Governing Nucleophilicity
1) More basic molecules tend to be more nucleophilic
when the nucleophilic atoms are of comparable size:
H
O
O
H
H
Poor nucleophile
Good nucleophile
pKa of conjugate acid = –1.5
pKa of conjugate acid = 15.5
2) Larger atoms (those lower on the periodic table) make
better nucleophiles:
H
O
H
H
Weaker nucleophile
S
H
Stronger nucleophile
Electrophiles in the Molecules of Life
O
O
O
N
H
Peptide hydrolysis
O
Translation
O
P O
O
DNA polymerization
DNA hydrolysis
Protein phosphorylation
• The most common electrophiles in the chemistry of life are
C=O and P=O
• Groups that are more electron-poor are more electrophilic
and therefore react more quickly with nucleophiles
4
Acids and Bases Can Enhance
Electrophilicity and Nucleophilicity
H
more electron-rich
BASE
:OH-
O
O
H
H
Better nucleophile
H
N
H
N
ACID
H+
more electron-poor
O
O
H
Better electrophile
Base Catalysis of Amide Hydrolysis
H
N
O
H
O
H
N
!" O
H
H
N
O H
H
O
H
H
N
!"
O
Weaker nucleophile:
slower reaction,
higher ΔG‡
!+
O
H2O + base
‡
O H
‡
Better nucleophile:
faster reaction,
lower ΔG‡
δ−
• Base can accelerate amide hydrolysis by deprotonating
water, increasing its nucleophilicity
5
Base Catalysis by HIV Protease
H
N
O
O
H
H
N
deprotonation
of water
H
O
O
O
!" O
O H
H
H
O
O
O
Asp 25
of HIV protease
O H
δ−
H
O
H
O
‡
H
N
O
O
O
H
O
O
Asp 25
of HIV protease
• HIV protease precisely positions Asp 25 to serve as a base
to deprotonate water
• The enhanced nucleophilicity of deprotonated water
accelerates amide hydrolysis
Acid Catalysis of Amide Hydrolysis
‡
H
N
O
H
N
H
O
!" O
H
amide
+
acid
H
H
N
H
N
O
H
O
H
H
O H
δ+
O
δ+
H
O H
δ+
‡
Weaker electrophile:
slower reaction,
higher ΔG‡
Stronger electrophile:
faster reaction,
lower ΔG‡
H
• Acid can accelerate amide hydrolysis by protonating the
amide oxygen, increasing its electrophilicity
6
Acid Catalysis by HIV Protease
H
N
‡
H
N
protonation
of amide
O
O
H
!" O
H
H
N
O H
H O
δ−
O H
H
O
O
H
O
O
O
Asp 25’
of HIV
protease
O
H
O
O
O
O
H
O
O
Asp 25’
of HIV
protease
• HIV protease precisely positions Asp 25’ to serve as an
acid to protonate the substrate amide, increasing its
electrophilicity and accelerating amide hydrolysis
Enzymes Can Catalyze Reactions in Ways
That Simple Acids and Bases Cannot
H
N
O
O
H
N
O
H
H
H
How can an acid and base
simultaneously catalyze a
single reaction?
H
H
O
BA
:O SE
H-
ID
AC +
H
O
O
enzyme acid
O
O
enzyme base
• Enzymes can simultaneously use acidic and basic groups
that, in a flask, would wander and neutralize each other
7
HIV Protease Active Site in Action
attacking
water
Ile 50’
H
substrate
Asp 25’
Asp 25
Note: locations of hydrogen atoms are usually inferred (and often not shown)
HIV Protease Catalysis Summary
Proximity and
orientation effects
H
N
O
H
H
O
O
H
O
Tetrahedral
intermediate
H
N
O H
H O
O
O
‡
H
N
δ−O H
δ−
H
O
O
O H
O
O
O
Asp 25’
Asp 25
Acid
catalysis
H
O
δ−O
O
O
H
O
‡
O
O H N
O H
O
H
H
O
δ− O
O
O
Base
catalysis
O
Base
catalysis
H
N
H O
O
H
O
H
H
N
H
H
O
O
O
O
Acid
catalysis
8
Lectures 15-16: The Molecular Basis of Enzyme Catalysis: HIV Protease
1.
The function and structure of HIV protease
a.
Introduction to proteases
b.
Discovery of HIV protease
c.
Overview of the three-dimensional structure of HIV protease
Chemical reactions and the energies driving them
a.
Amide bond cleavage: the reaction catalyzed by HIV protease
b.
Thermodynamics of a reaction and free energy
c.
Kinetics of a reaction and ΔG‡
2.
d.
Reaction energy diagrams
e.
Transition states, intermediates, and how to draw them
How enzymes accelerate chemical reactions: the case of HIV protease
a.
Catalysts alter a reaction’s kinetics, but not its thermodynamics
b.
Chemical strategies behind enzyme catalysis
i.
Proximity and orientation effects
ii.
Nucleophilicity and electrophilicity
iii.
Acid and base catalysis
The molecular basis of substrate specificity
a.
Trypsin substrate specificity
b.
HIV protease substrate specificity
3.
4.
Protease Substrate Specificity
Scissile
bond
S1
S3
O
H
N
O
P4
P3
N
H
S4
To N-terminal end
of substrate
O
H
N
O
P2
S2
P1
N
H
H
N
O
S2’
O
P1'
P2'
N
H
O
S1’
Enzyme specificity
pockets recognize the
specific amino acid
residues surrounding the
bond to be hydrolyzed
S4’
H
N
O
P3'
P4'
N
H
H
N
O
S3’
To C-terminal end
of substrate
9
Trypsin: A Digestive Protease that Cleaves
Substrates Containing Lys or Arg
substrate peptide
trypsin
enzyme
enzyme
Asp189
S1
substrate
Lys P1
+
–
scissile bond
trypsin active
site residues
Basis of Trypsin Substrate Specificity
Asp 189
S1
Asp 189
S1
O
H2N
O
O
O
NH2
H3N
NH
N
H
H
N
O
Arg
P1
N
H
H
N
Lys
P1
O
• The presence of anionic Asp 189 in the S1 site causes a
strong preference for P1 to be a cationic Lys or Arg
10
HIV Protease-Substrate Interactions
Animation rendered by Brian Tse
The HIV Protease Active
Site: A Closer Look
Note: structure is
of an inactive
Asn 25 mutant of
HIV protease
complexed with
a substrate.
11
HIV Protease Substrate Selectivity
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
• HIV protease recognizes more substrate amino acids than
trypsin, but without a strong preference at any one position
HIV Protease Specificity: P1 and P3
water
S3
H
H
O
H
N
H
N
NH
H
HN
Arg P3
H
N
O
• HIV protease’s
structure can explain
some aspects of its
substrate specificity
H
H
N
O
N
H
O
Gly 49
S1
O
H
N
N
H
O
Leu P1
O
N
H
H
N
O
• P1= large and
hydrophobic;
complements
S1 = Gly 49
• P3 = polar orAsn 25’
charged; S3 contains
a bound water
12
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 strategies
to create a precise transition state-stabilizing active site
environment
• Enzymes use proximity and orientation effects to increase
the concentration of substrates, increasing rates of reactions
• Enzymes use acid and base catalysis to enhance the
nucleophilicity or electrophilicity of reactants
• Specificity arises from protein-substrate interactions
O
O
O
HN
N
S
NH
N
N
HO
OH
S
N
Life Sciences 1a
Lecture Slides Set 10
Fall 2006-2007
Prof. David R. Liu
13
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
Lecture Readings
Required: Lecture Notes
McMurray p. 808-810, 640-642
Impact of Anti-HIV Drugs
• 1990s: anti-HIV drugs transform HIV infection from a short
death sentence to a chronic (but very serious) illness
• 13 FDA-approved drugs inhibit HIV reverse transcriptase; 9
drugs 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
14
Drug Development is Very Difficult
• Total cost to develop a drug = ~$1 billion + ~10-15 years
Successful Drugs Must Satisfy Many
Chemical and Biological Requirements
1) Potency (affinity)
Keq = Ka = 1÷Kd
+
drug
protein target
drug-protein complex
2) Specificity (toxicity, immunogenicity)
non-target
target
non-target non-target
3) Bioavailability
oral
cellular
4) Biostability
k
inactive or toxic
5) Economics
15