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

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

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

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

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

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

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

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