The insert within the catalytic domain of tripeptidyl-peptidase II is important for the formation of the active complex.
Tripeptidyl-peptidase II (TPP II) is a large (Mr>10(6)) tripeptide-releasing enzyme with an active site of the subtilisin-type. Compared with other subtilases, TPP II has a 200 amino-acid insertion between the catalytic Asp44 and His264 residues, and is active as an oligomeric complex. This study demonstrates that the insert is important for the formation of the active high-molecular mass complex. A recombinant human TPP II and a murine TPP II were found to display different complex-forming characteristics when over-expressed in human 293-cells; the human enzyme was mainly in a nonassociated, inactive state whereas the murine enzyme formed active oligomers. This was surprising because native human TPP II is purified from erythrocytes as an active oligomeric complex, and the amino-acid sequences of the human and murine enzymes were 96% identical. Using a combination of chimeras and a single point mutant, the amino acid responsible for this difference was identified as Arg252 in the recombinant human sequence, which corresponds to a glycine in the murine sequence. As Gly252 is conserved in all sequenced variants of TPP II, the recombinant enzyme with Arg252 is atypical. Nevertheless, as Arg252 evidently interferes with complex formation, and this residue is close to the catalytic His264, it may also explain why oligomerization influences enzyme activity. The exact mechanism for how the G252R substitution interferes with complex formation remains to be determined, but will be of importance for the understanding of the unique properties of TPP II.
Tomkinson
Birgitta
B
Department of Biochemistry, Uppsala University, Biomedical Center, Uppsala, Sweden. Birgitta.Tomkinson@biokem.uu.se
Ní Laoi
Bairbre
B
Wellington
Kimberly
K
D000595:Amino Acid Sequence; D000626:Aminopeptidases; D000818:Animals; D020134:Catalytic Domain; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000737:chemistry*
D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C048001:tripeptidyl-peptidase 2; D012697:Serine Endopeptidases