Mammalian glutaminyl cyclases and their isoenzymes have identical enzymatic characteristics.
Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate residues at the N-terminus of several peptides and proteins from plants and animals. Recently, isoenzymes of mammalian QCs have been identified. In order to gain further insight into the biochemical characteristics of isoQCs, the human and murine enzymes were expressed in the secretory pathway of Pichia pastoris. Replacement of the N-terminal signal anchor by an alpha-factor prepropeptide from Saccharomyces cerevisiae resulted in poor secretion of the protein. Insertion of an N-terminal glycosylation site and shortening of the N-terminus improved isoQC secretion 100-fold. A comparison of different recombinant isoQC proteins did not reveal an influence of mutagenic changes on catalytic activity. An initial characterization showed identical modes of substrate conversion of human isoQC and murine isoQC. Both proteins displayed a broad substrate specificity and preference for hydrophobic substrates, similar to the related QC. Likewise, a determination of the zinc content and reactivation of the apo-isoQC revealed equimolar zinc present in QC and isoQC. Far-UV CD spectroscopic analysis of murine QC and isoQC indicated virtually identical structural components. The present investigation provides the first enzymatic characterization of mammalian isoQCs. QC and isoQC represent very similar proteins, which are both present in the secretory pathway of cells. The functions of QCs and isoQC probably complement each other, suggesting a pivotal role of pyroglutamate modification for protein and peptide maturation.
Probiodrug AG, Halle/Saale, Germany.
Anett
A
Stephan
Michael
M
Wermann
Alex
A
von Bohlen
Birgit
B
Koch
Holger
H
Cynis
Hans-Ulrich
HU
Demuth
Stephan
S
Schilling
D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002942:Circular Dichroism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D007527:Isoenzymes; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase