Chemical modification of dipeptidyl peptidase iv: involvement of an essential tryptophan residue at the substrate binding site.
Inactivation of pig kidney dipeptidyl peptidase IV (EC 3.4.14.5) by photosensitization in the presence of methylene blue at pH 7.5 was observed to have pseudo-first-order kinetics. During the process, until over 95% inactivation was achieved, the histidine and tryptophan residues were decreased from 14.0 to 2.7 and 12.6 to 7.1, respectively, per 94,000-Da subunit, without any detectable changes in other photosensitive amino acids. Modification of four histidine residues per subunit using diethylpyrocarbonate resulted in only 30% inactivation of the enzyme, while N-bromosuccinimide almost completely inactivated the enzyme with the modification of only one tryptophan residue per subunit, as determined by absorption spectrophotometry at 280 nm. The protective action of the substrate and inhibitors such as Ala-Pro-Ala and Pro-Pro against the modification of tryptophan residues with N-bromosuccinimide was observed both fluorometrically and by measurement of activity. On the basis of these results it is suggested that one of the tryptophan residues in the enzyme subunit is essential for the functioning of the substrate binding site of pig kidney dipeptidyl peptidase IV.
D000818:Animals; D001665:Binding Sites; D001974:Bromosuccinimide / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000037:antagonists & inhibitors* / Q000528:radiation effects; D006639:Histidine / Q000528:radiation effects; D007700:Kinetics; D008027:Light; D010777:Photochemistry; D011480:Protease Inhibitors; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013552:Swine; D014364:Tryptophan / Q000528:radiation effects
D011480:Protease Inhibitors; D006639:Histidine; D014364:Tryptophan; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4; D001974:Bromosuccinimide; D004047:Diethyl Pyrocarbonate