IDENTIFICATION OF THE ACTIVE-SITE RESIDUES IN DIPEPTIDYL PEPTIDASE-IV BY AFFINITY LABELING AND SITE-DIRECTED MUTAGENESIS

The active site of dipeptidyl peptidase IV (DPPIV) was examined by chemical modification and site-directed mutagenesis. Purified DPPIV was covalently modified with [H-3]diisopropyl fluorophosphate (DFP). The radiolabeled DPPIV was digested with lysyl endopeptidase, and the peptides were separated by high-performance liquid chromatography. A single H-3-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 [H-3]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.