Activation mechanism of pro-astacin: role of the pro-peptide, tryptic and autoproteolytic cleavage and importance of precise amino-terminal processing.
Astacin (EC 3.4.24.21) is a prototype for the astacin family and for the metzincin superfamily of zinc peptidases, which comprise membrane-bound and secreted enzymes involved in extracellular proteolysis during tissue development and remodelling. Generally, metzincins are translated as pro-enzymes (zymogens), which are activated by removal of an N-terminal pro-peptide. In astacin, however, the mode of zymogen activation has been obscured, since the pro-form does not accumulate in vivo. Here we report the detection of pro-astacin in midgut glands of brefeldin A-treated crayfish (Astacus astacus) by immunoprecipitation and mass spectrometry. We demonstrate that the pro-peptide is able to shield the active site of mature astacin as a transient inhibitor, which is degraded slowly. In vitro studies with recombinant pro-astacin in the absence of another protease reveal a potential of auto-proteolytic activation. The initial cleavage in this autoactivation appears to be an intramolecular event. This is supported by the fact that the mutant E93A-pro-astacin is incapable of autoactivation, and completely resistant to cleavage by mature astacin. However, this mutant is cleaved by Astacus trypsin within the pro-peptide. This probably reflects the in vivo situation, where Astacus trypsin and astacin work together during pro-astacin activation. In a first step, trypsin produces amino-terminally truncated pro-astacin derivatives. These are trimmed subsequently by each other and by astacin to yield the mature amino terminus, which forms a salt-bridge with Glu103 in the active site. The disruption of this salt-bridge in the mutants E103A and E103Q results in extremely heat labile proteins, whose catalytic activities are not altered drastically, however. This supports a concept according to which the linkage of Glu103 to the precisely trimmed amino terminus is a crucial structural prerequisite throughout the astacin family.
Yiallouros
Irene
I
Institute of Zoophysiology, University of Münster, Hindenburgplatz 55, Munster, Germany.
Kappelhoff
Reinhild
R
Schilling
Oliver
O
Wegmann
Frank
F
Helms
Mike W
MW
Auge
Astrid
A
Brachtendorf
Gertrud
G
Berkhoff
Eva Grosse
EG
Beermann
Bernd
B
Hinz
Hans Jürgen
HJ
König
Simone
S
Peter-Katalinic
Jasna
J
Stöcker
Walter
W
D000818:Animals; D003400:Astacoidea / Q000201:enzymology*; D001665:Binding Sites; D004064:Digestive System / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D009154:Mutation; D010447:Peptide Hydrolases / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014357:Trypsin / Q000494:pharmacology*
D004792:Enzyme Precursors; D010455:Peptides; D010447:Peptide Hydrolases; D014357:Trypsin; D008666:Metalloendopeptidases; C071288:astacin