Identification of the active site residues in dipeptidyl peptidase IV by affinity labeling and site-directed mutagenesis

• Published in: Biochemistry (Easton) 1992-03, Vol.31 (9), p.2582-2587
• Main Authors: Ogata, Shigenori, Misumi, Yoshio, Tsuji, Emiko, Takami, Noboru, Oda, Kimimitsu, Ikehara, Yukio
• Format: Article
• Language: English
• Subjects: active sites; Affinity Labels; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Base Sequence; Binding Sites; Biological and medical sciences; Cell Line; Chromatography, High Pressure Liquid; Dipeptidyl Peptidase 4; dipeptidyl peptidase IV; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - chemistry; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - genetics; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - metabolism; DNA - chemistry; DNA - genetics; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; identification; Isoflurophate; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments - isolation & purification; Peptide Fragments - metabolism; Plasmids; Serine Endopeptidases - metabolism; site-directed mutagenesis; Transfection; Tritium
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00124a019

The active site of dipeptidyl peptidase IV (DPPIV) was examined by chemical modification and site-directed mutagenesis. Purified DPPIV was covalently modified with [3H]diisopropyl fluorophosphate (DFP). The radiolabeled DPPIV was digested with lysyl endopeptidase, and the peptides were separated by high-performance liquid chromatography. A single 3H-containing peptide was obtained and analyzed for amino acid sequence and radioactivity distribution. A comparison of the determined sequence with the predicted primary structure of DPPIV [Ogata, S., Misumi, Y., & Ikehara, Y. (1989) J. Biol. Chem. 264, 3596-3601] revealed that [3H]DFP was bound to Ser631 within the sequence Gly629-Trp-Ser-Tyr-Gly633, which corresponds to the consensus sequence Gly-X-Ser-X-Gly proposed for serine proteases. To further identify the essential residues in the active-site sequence, we modified the DPPIV cDNA by site-directed mutagenesis to encode its variants. Expression of the mutagenized cDNAs in COS-1 cells demonstrated that any single substitution of Gly629, Ser631, or Gly633 with other residues resulted in the complete loss of the enzyme activity and DFP binding. Although substitution of Trp630---Glu or Tyr632---Phe caused no effect on the enzyme activity, that of Tyr632---Leu or Gly abolished the activity. These results indicate that the sequence Gly-X-Ser-(Tyr)-Gly is essential for the expression of the DPPIV activity.