Aspartate 496 from the subsite S2 drives specificity of human dipeptidyl peptidase III

Area of Science:

• Biochemistry
• Enzymology
• Proteolysis

Background:

• Human dipeptidyl peptidase III (hDPP III) is a metallopeptidase involved in protein catabolism and oxidative stress.
• Understanding hDPP III substrate specificity is key to elucidating its biological roles.

Purpose of the Study:

• To investigate the structural basis for hDPP III's preference for diarginyl arylamide substrates.
• To identify key residues determining substrate specificity using site-directed mutagenesis.

Main Methods:

• Site-directed mutagenesis was employed to alter the S2 subsite of hDPP III.
• Kinetic studies were performed to analyze enzyme-substrate interactions.
• Binding assays were conducted using peptides and tynorphin.

Main Results:

• The D496G mutant exhibited significantly reduced substrate selectivity, indicated by an increased Km value.
• The D496G mutant showed decreased binding affinity for N-terminal arginine peptides and tynorphin.
• The S504G mutation did not significantly impact substrate binding or selectivity.

Conclusions:

• Asp496 is identified as a critical determinant of human dipeptidyl peptidase III substrate specificity.
• The S2 subsite plays a crucial role in the enzyme's preference for specific substrates, particularly those with N-terminal arginine.