Continuous spectrometric assays for glutaminyl cyclase activity.
The enzymatic conversion of one chromogenic substrate, l-glutamine-p-nitroanilide, and two fluorogenic substrates, l-glutaminyl-2-naphthylamide and l-glutaminyl-4-methylcoumarinylamide, into their respective pyroglutamic acid derivatives by glutaminyl cyclase (QC) was estimated by introducing a new coupled assay using pyroglutamyl aminopeptidase as the auxiliary enzyme. For the purified papaya QC, the kinetic parameters were found to be in the range of those previously reported for other glutaminyl peptides, such as Gln-Gln, Gln-Ala, or Gln-tert-butyl ester. The assay can be performed in the presence of ammonia up to a concentration of 50 mM. Increasing ionic strength, e.g., potassium chloride up to 300 mM, resulted in an increase in enzymatic activity of about 20%. This is the first report of a fast, continuous, and reliable determination of QC activity, even in the presence of ammonium ions, during the course of protein purification and enzymatic analysis.
Schilling
Stephan
S
Laboratory of Biochemistry, probiodrug AG, Weinbergweg 22, 06120 Halle/Saale, Germany.
Hoffmann
Torsten
T
Wermann
Michael
M
Heiser
Ulrich
U
Wasternack
Claus
C
Demuth
Hans-Ulrich
HU
D019881:Aminoacyltransferases / Q000032:analysis* / Q000378:metabolism; D029441:Carica / Q000201:enzymology; D002863:Chromogenic Compounds / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods*; D011751:Pyroglutamyl-Peptidase I / Q000032:analysis; D013053:Spectrophotometry
D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase; D011751:Pyroglutamyl-Peptidase I