Solid-phase active site inhibitors of proteinases.

Area of Science:

• Biochemistry
• Enzyme Inhibitors
• Solid-Phase Chemistry

Background:

• Proteolytic enzymes like chymotrypsin and trypsin play crucial roles in biological processes.
• Developing specific inhibitors is essential for studying enzyme function and for therapeutic applications.
• Solid-phase synthesis offers advantages for enzyme inhibition and purification.

Purpose of the Study:

• To synthesize and characterize solid-phase inhibitors for chymotrypsin-like and trypsin-like enzymes.
• To evaluate the efficacy of these immobilized inhibitors in enzyme inactivation and binding.
• To explore the potential of solid-phase inhibitors for isolating specific serine proteases and determining active-site sequences.

Main Methods:

• Synthesis of phenylalanine chloromethyl ketone immobilized on porous glass beads.
• Synthesis of Sepharose-bound lysine chloromethyl ketones.
• Enzyme inhibition and binding assays with immobilized reagents.
• Cyanogen bromide cleavage and enzymatic digestion of immobilized enzymes.

Main Results:

• Phenylalanine chloromethyl ketone-porous glass inhibited 20 nmol of chymotrypsin and bound 30 nmol of protein per gram of glass.
• Sepharose-bound methionyllysyl chloromethyl ketone inactivated and bound approximately 6.8 nmol of enzyme per ml of settled gel.
• Preliminary experiments indicated the feasibility of releasing peptides while retaining the active-site histidine-containing peptide.

Conclusions:

• Solid-phase active site-directed inhibitors can be effectively synthesized for serine proteases.
• Immobilized inhibitors demonstrate significant enzyme inhibition and binding capacity.
• This solid-phase approach holds promise for the specific isolation of serine proteases and for sequencing residues around the active-site histidine.