Transcript Chapter 6

Enzyme Rate Enhancement
How do enzymes work to catalyze reactions?
Enzyme Classes by Reaction Type
Oxidation-Reduction Reaction:
Oxidoreductase
Functional Group Transfer: Transferase
Cutting with Water: Hydrolysis
Group Elimination with Double Bond
Formation: Lyase
Compound Conversion to an Isomeric
Form: Isomerase
Triose phosphate isomerase
Bond Formation with ATP Hydrolysis:
Ligase
DNA Ligase -- nucleotide base coupling with loss of PPi
Chymotrypsin Catalyzes Peptide Hydrolysis
Enzyme specificity in terms of:
Site of hydrolysis and
Substrate reactivity
How can a reaction be pushed in the forward direction?
Reactions that are Spontaneous in
the Forward and Reverse Direction
How do enzymes affect the energy diagram of a reaction?
Enzymes Decrease the Activation Energy
How can
enzymes
lower the
transition
state?
Is external
energy
required?
Mechanisms of Enzyme Catalysis
Acid-base, covalent, metal ion, orientation
and proximity
Ketone-Enol Conversion
What mechanism of enzyme catalysis is operative here?
Amino Acid
Residues that
Function in
Acid-Base
Catalysis
Conventions for
Depicting
Reaction
Mechanisms
Curved arrow indicates
electron rearrangement
during a reaction
Covalent Catalysis
A formal positive charge favors electron flow to the
nitrogen group
Covalent Catalysis
Amino Acids that can Participate in
Covalent Catalysis
Metal Ion Catalysis
Components that Facilitate Enzyme Catalysis
What is the cofactor in ATP hydrolysis?
What is the co-substrate in an oxidation-reduction reaction?
Enzymes Decrease the Activation Energy
Mechanisms of Enzyme Catalysis
• Acid-Base Catalysis
• Covalent Catalysis
• Metal Ion Catalysis
• Orientation/Proximity
Effects
• Preferential
Transition- State
Binding
Proximity and Orientation Effects
Facilitate Catalysis
Chymotrypsin Specificity
Cleaves peptides on the C-terminus side of
hydrophobic residues (e.g. Phe, Tyr and Try)
Active Site Mapping via Irreversible Inhibitors
Diisopropylphosphofluoridate (DIPF) inhibits
chymotrypsin by modifying 1 of 28 serine residues
Active Site Mapping via Irreversible Inhibitors
Covalent Catalysis for Chymotrypsin: a
Two Step Process
• Enzyme acylation with leaving group departure
• Enzyme deacylation
What is the leaving group with Chymotrypsin?
Chymotrypsin Catalysis Proceeds
via a Two-Step Mechanism
Chromogenic substrate
for kinetic studies
Why is this compound
not an ideal substrate
mimic?
Chymotrypsin Catalytic Triad
• Catalytic triad serves as the site of catalysis
• Aspartate and histidine contribute serine’s basicity
• Serine serves as a nucleophile in covalent catalysis
What type of catalysis occurs?
Mechanism of Peptide Hydrolysis in
Chymotrypsin
Substrate binding via
nucleophilic attack
Mechanism of Peptide Hydrolysis in
Chymotrypsin
Polypeptide original C-side
serves as leaving group
Mechanism of Peptide Hydrolysis in
Chymotrypsin
Water attacks original Nside of polypeptide
Mechanism of Peptide Hydrolysis in
Chymotrypsin
Polypeptide original N-side
serves as leaving group
and enzyme is regenerated
Chymotrypsin Hydrolysis
Tetrahedral-Intermediate
Stabilization in Chymotrypsin
H-bonds ideally positioned
in the oxyanion hole stabilize
the sp3 transition state
Chymotrypsin Specificity Pocket
Large structural pocket
lined with hydrophobic
amino acids favors bulky
hydrophobic residues
Serine Proteases Differ in Little
Except Their Specificity
Pockets
Chymotrypsin
Trypsin
Elastase
Substrate Specificity
Observed with each
Proteolytic Enzyme
• Papain cleave peptides
non-selectively
• Trypsin cleaves carboxyl
side of bulky + charged Rgroups
• Chymotrypsin cleaves
carboxyl side of bulky
aromatic R-groups
Thrombin
Divergent Evolution
Percent Sequence Identity among Three Serine Proteases
Bovine trypsin
Bovine chymotrypsinogen
Porcine elatase
Common ancestor with
retention of overall structure
and catalytic mechanism
100%
53%
48%
Convergent Evolution
Bovine versus bacterial serine protease
No sequence or structural similarity but the same
catalytic triad and oxyanion hole in the active site
Enzyme-Substrate
Binding Critical for
Catalysis
Lock and Key Model
Enzyme Active Site
• 3-D cleft or crevice
• Small part of enzyme
• Unique micro-environment
• Substrate binding by weak
forces
Induced Fit Model
Substrate-Induced Enzyme Conformational
Change
Substrate (-)
Substrate (+)
Inhibition by Transition State Analogs
Pyrrolidine the natural
substrate binds 160 less
tightly than pyrrole a
transition state analog.
What is the favored
enzyme binding
geometry?
Rate of Enzyme Catalysis
Explain why enzyme activity increases with
temperature and then precipitously drops off
RNAas A Digestive Enzyme Cleaving
Mechanism
Why does
ribonuclease
catalyzes the
hydrolysis of RNA
but not DNA
Conversion of Adenosine to Inosine
What does the
much greater
binding
affinity of 1,6dihydropurine
ribonucleoside
than the
substrate
indicate about
the enzyme
mechanism?
Chapter 6 Problems: 1, 3, 7, 9, 11, 15, 20,
23, 25 and 37