Substrate specificity of glutaminyl cyclases from plants and animals.
Glutaminyl cyclases (QC) catalyze the intramolecular cyclization of N-terminal glutamine residues of peptides and proteins. For a comparison of the substrate specificity of human and papaya QC enzymes, a novel continuous assay was established by adapting an existing discontinuous method. Specificity constants (kcat/Km) of dipeptides and dipeptide surrogates were higher for plant QC, whereas the selectivity for oligopeptides was similar for both enzymes. However, only the specificity constants of mammalian QC were dependent on size and composition of the substrates. Specificity constants of both enzymes were equally pH-dependent in the acidic pH-region, revealing a pKa value identical to the pKa of the substrate, suggesting similarities in the substrate conversion mode. Accordingly, both QCs converted the L-beta homoglutaminyl residue in the peptide H-beta homoGln-Phe-Lys-Arg-Leu-Ala-NH2 and the glutaminyl residues of the branched peptide H-Gln-Lys(Gln)-Arg-Leu-Ala-NH2 as well as the partially cyclized peptide H-Gln-cyclo(N epsilon-Lys-Arg-Pro-Ala-Gly-Phe). In contrast, only QC from C. papaya was able to cyclize a methylated glutamine residue, while this compound did not even inhibit human QC-catalysis, suggesting distinct substrate recognition pattern. The conversion of the potential physiological substrates [Gln1]-gastrin, [Gln1]-neurotensin and [Gln1]-fertilization promoting peptide indicates that human QC may play a key role in posttranslational modification of most if not all pGlu-containing hormones.
Schilling
Stephan
S
Probiodrug AG, Weinbergweg 22, D-06120 Halle/Saale, Germany.
Manhart
Susanne
S
Hoffmann
Torsten
T
Ludwig
Hans-Henning
HH
Wasternack
Claus
C
Demuth
Hans-Ulrich
HU
D000596:Amino Acids / Q000737:chemistry; D019881:Aminoacyltransferases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D029441:Carica / Q000201:enzymology*; D002384:Catalysis; D002417:Cattle; D003500:Cyclization; D004151:Dipeptides / Q000378:metabolism; D004795:Enzyme Stability / Q000187:drug effects; D005755:Gastrins / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009496:Neurotensin / Q000378:metabolism; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D009994:Osmolar Concentration; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013973:Thyrotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000378:metabolism
D000596:Amino Acids; D004151:Dipeptides; D005755:Gastrins; D007656:Ketoglutaric Acids; D009842:Oligopeptides; D011994:Recombinant Proteins; D005973:Glutamine; D009243:NAD; D007987:Gonadotropin-Releasing Hormone; D009496:Neurotensin; D013973:Thyrotropin-Releasing Hormone; C059697:pyroglutamyl-glutamyl-proline amide; D005969:Glutamate Dehydrogenase; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase; D011761:Pyrrolidonecarboxylic Acid