the enzyme is translated as an inactive single chain precursor, being post-translationally modified into an active enzyme. The post-translational modification takes place in two steps. The first intramolecular autocatalytic proteolysis takes place at one end of the spacer peptide by a nucleophilic Ser or Thr, which in turn becomes a new N-terminal Ser or Thr. The second intermolecular modification cleaves off the other end of the spacer peptide by another enzyme molecule
the enzyme is processed in two sequential steps of intramolecular autoproteolysis involving two distinct proteolytic mechanisms, the first mediatzed by a serine residue and the second by a glutamate
a first autoproteolytic cleavage site of the pro-enzyme is G239-S240, a second cleavage site is A232-S233, determined by tandem MS/MS analysis of the purified alpha-subunit C-terminus
the enzyme is synthesized as a single folded precursor protein made of 782 amino acid residues that, once folded, undergoes an autocatalytic processing event to produce the mature alpha/beta heterodimer, an internal linker segment of ten residues (GDASDAAGGG) is removed from the pre-enzyme between the final alpha- and beta-chains, maturation process, overview
the gene encoding the enzyme is expressed as a precursor polypeptide consisting of a signal peptide followed by alpha- and beta-subunits, which are separated by a spacer peptide. Removing the signal peptide has little effect on precursor processing or enzyme activity. The precursor is supposed to be processed autocatalytically, probably intramolecularly
activation of precursor consists of primary and secondary autoproteolytic cleavages, generating a terminal residue with both a nucleophile and a base and releasing a nine amino acid spacer peptide. Precursor activation is likely triggered by conformational constraints within the spacer peptide, probably inducing a peptide flip. Autoproteolytic site solvent molecules, which have been trapped in a hydrophobic environment by the spacer peptide, may play a role as a general base for nucleophilic attack. The activation results in building up a catalytic triad composed of Ser170/His192/Glu624. The triad is not linked to the usual hydroxyl but the free R-amino group of the N-terminal serine residue of the native enzyme. Stabilization of a transient hydroxazolidine ring during autoproteolysis would be critical during the N to O acyl shift