Converting trypsin to chymotrypsin: residue 172 is a substrate specificity determinant

Area of Science:

• Enzymology
• Protein engineering
• Biochemistry

Background:

• Trypsin and chymotrypsin share similar structures but differ in substrate specificity.
• Understanding these differences is key to enzyme engineering.

Purpose of the Study:

• To investigate the structural determinants of substrate specificity in trypsin and chymotrypsin.
• To engineer trypsin to exhibit chymotrypsin-like substrate specificity.

Main Methods:

• Site-directed mutagenesis was used to alter trypsin's amino acid residues.
• Specific mutations targeted the substrate binding pocket and surface loops.
• Enzyme activity assays were performed to assess substrate specificity.

Main Results:

• Mutating only the substrate binding pocket did not confer chymotrypsin-like specificity.
• Changes in surface loops combined with S1 site modifications yielded partial chymotrypsin-like activity.
• A Tyr172 to Trp mutation further enhanced activity, indicating synergistic interactions.

Conclusions:

• Substrate specificity is primarily determined by the catalytic processing rate, not solely substrate binding.
• Surface loops and specific residues like Tyr172 play crucial roles in defining enzyme specificity.
• Protein engineering can modulate enzyme specificity through targeted mutations.