1. Chap 9. Active-Site-Directed and Enzyme-Activated Irreversible Inhibitors: “Affinity Labels” and “Suicide Inhibitors”
Covalent modification of enzyme may cause a loss of enzyme activity by
blocking a catalytically essential residue
sterically Impeding substrate binding
distorting the protein or impairing the mobility of the protein
Specially designed irreversible inhibitors: binding to the active site

2. A. Chemical modification of proteins
Objectives
to modify residues in proteins chemically
to provide mechanistic information
to produce useful alterations of activity
Spectroscopic probes (reporter groups): to examine local structure or overall conformation
Ex. fluorescent derivatives, spin labels, nitration
The cross-linking of proteins: to measure the degree of association or subunit composition of oligomers
The insertion of radioactive tags
Searching for unusually reactive groups
Measuring the degree of exposure of groups to solvent
Measuring the pKa and ionic states of reactive groups
Assessing catalytically important group
Irreversibly inhibiting activity

3. The Reactive Groups in Proteins are Nucleophiles
-OH of Ser, Thr, and Tyr
ε-NH2 of Lys and α-NH2 of the N-termini
The imidazole ring of His
-S- of Cys
-CO2- of Asp, Glu, and C-termini
The majority reagents for modifying proteins: Electrophiles
Activated acyl compounds
Alkylating agents

4. B. Active-Site-Directed Irreversible Inhibitors (Affinity Label)
Designed to resemble a substrate
Binding specifically to the active site
Forming covalent bonds
Useful for identifying catalytically important residues and for determining the pKa
E + I E•I E-I KI kI
Specific binding
Unusual rapid reaction

5. The Irreversible Inhibition has Four Characteristic Features
Competitive inhibition by substrates
pH dependence
1:1 stoichiometry
Saturation kinetics obeyed
Examples
TPCK(tos-L-phenylalanine chloromethyl ketone): highly reactive
1,2-anhydro-D-mannitol 6-phosphate: revealing an unexpected catalytic residue
Diazoacetyl derivative of an amino acid ester: labeling un-ionized carboxyl group
Photoaffinity label: useful in mapping out residues at the active sites of enzyme and the binding sites of proteins

6. C. Enzyme-Activated Irreversible Inhibitors
Suicide Inhibitors, kcat inhibitors, mechanism-based inhibitors, trojan-horse inhibitors, enzyme-activated substrate inhibitors
Highly specific irreversible inhibitors – potential therapeutic agents
Unreactivity remaining without enzyme
Activated by the target enzyme
kI >> kdiss
Based on the generation of an reactive intermediate: conjugated double bonds
Acetylenic compounds, β,γ-unsaturated compounds, β-halo compounds

7. 1. Pyridoxal phosphate-linked enzymes
2. Monoamine oxidases and flavoproteins

8. D. Slow, Tight-Binding Inhibition
A slow onset of inhibition:
slow conformational change in the enzyme from a weak binding to a tight binding state