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 [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.
Department of Biochemistry, Fukuoka University School of Medicine, Japan.
S
S
Ogata
Y
Y
Misumi
E
E
Tsuji
N
N
Takami
K
K
Oda
Y
Y
Ikehara
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry / Q000235:genetics; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007531:Isoflurophate; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D012697:Serine Endopeptidases / Q000378:metabolism; D014162:Transfection; D014316:Tritium
D000345:Affinity Labels; D010446:Peptide Fragments; D014316:Tritium; D007531:Isoflurophate; D004247:DNA; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase