Cephalosporin acylase (CA), a member of the N-terminal nucleophile hydrolase family, is activated through sequential primary and secondary autoproteolytic reactions with the release of a pro segment. We have determined crystal structures of four CA mutants. Two mutants are trapped after the primary cleavage, and the other two undergo secondary cleavage slowly. These structures provide a look at pro-segment conformation during activation in N-terminal nucleophile hydrolases. The highly strained helical pro segment of precursor is transformed into a relaxed loop in the intermediates, suggesting that the relaxation of structural constraints drives the primary cleavage reaction. The secondary autoproteolytic step has been proposed to be intermolecular. However, our analysis provides evidence that CA is processed in two sequential steps of intramolecular autoproteolysis involving two distinct residues in the active site, the first a serine and the second a glutamate.autoproteolysis ͉ precursor activation ͉ intermediate structure ͉ pro segment P osttranslational autoproteolysis is a mechanism used to activate many proteins via self-catalyzed peptide bond rearrangements, which play an essential role in a wide variety of biological processes. They include activation cascades such as blood coagulation and fibrinolysis, cell death, embryonic development, protein targeting and degradation, viral protein processing, and zymogen activation (1-6). Increasing evidence suggests that autoproteolysis may be involved in more biological activation processes than previously appreciated. These processes are all mediated by intramolecular or intermolecular autocleavage reactions. Four types of intramolecular protein modifications have been studied extensively: hedgehog proteins, inteins, N-terminal nucleophile (Ntn) hydrolases, and pyruvoyl enzymes (4,(7)(8)(9).Members of the Ntn hydrolase family have divergent sequences, but share an ␣␣ sandwich structural motif and a common enzyme mechanism (7,8). In addition, they catalyze internal peptide bond rearrangement through an N 3 O or N 3 S acyl shift by a nucleophilic Ntn amino acid created by autoproteolytic processing (4, 7). Structures of mature Ntn hydrolases, including cephalosporin acylase (CA), penicillin G acylase (PA), penicillin V acylase, glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase, 20S proteasome  subunit (PRO), glycosylasparaginase (GA), and L-aminopeptidase, have been determined (10-17). Structures of the precursors of CA, PA, PRO, and GA have been reported, providing some insight into the activation mechanism (11,(18)(19)(20)(21)(22). However, the understanding of mechanism cannot be complete without structural information on the first cleavage intermediates.Ntn hydrolases are of particular interest because they include acylase enzymes used in the biosynthesis of the -lactam antibiotics cephalosporin and penicillin. We have focused on CA. This enzyme is expressed as a 76-kDa precursor that is activated by two sequential autocleavage steps (11, 23) (Fig....
