Abstract
Cephalosporin acylase (CA) precursor is translated as a single polypeptide chain and folds into a self-activating pre-protein. Activation requires two peptide bond cleavages that excise an internal spacer to form the mature αβ heterodimer. Using Q-TOF LC-MS, we located the second cleavage site between Glu(159) and Gly(160), and detected the corresponding 10-aa spacer (160)GDPPDLADQG(169) of CA mutants. The site of the second cleavage depended on Glu(159): moving Glu into the spacer or removing 5-10 residues from the spacer sequence resulted in shorter spacers with the cleavage at the carboxylic side of Glu. The mutant E159D was cleaved more slowly than the wild-type, as were mutants G160A and G160L. This allowed kinetic measurements showing that the second cleavage reaction was a first-order, intra-molecular process. Glutaryl-7-aminocephalosporanic acid is the classic substrate of CA, in which the N-terminal Ser(170) of the β-subunit, is the nucleophile. Glu and Asp resemble glutaryl, suggesting that CA might also remove N-terminal Glu or Asp from peptides. This was indeed the case, suggesting that the N-terminal nucleophile also performed the second proteolytic cleavage. We also found that CA is an acylpeptide hydrolase rather than a previously expected acylamino acid acylase. It only exhibited exopeptidase activity for the hydrolysis of an externally added peptide, supporting the intra-molecular interaction. We propose that the final CA activation is an intra-molecular process performed by an N-terminal nucleophile, during which large conformational changes in the α-subunit C-terminal region are required to bridge the gap between Glu(159) and Ser(170).
Highlights
24476 JOURNAL OF BIOLOGICAL CHEMISTRY according to the SCOP and Pfam data bases, and belongs to the PB clan and S45 peptidase family according to the MEROPS data base [1,2,3]
The Second Autoproteolytic Cleavage of Cephalosporin acylase (CA) Is an Intra-molecular Interaction Catalyzed by Ntn—Due to the lack of information about conformational dynamics, the possibility that the second autoproteolytic cleavage of CA could be an intra-molecular interaction catalyzed by Ser170, the Ntn, has been neglected for years
Two mechanisms, an intra-molecular interaction catalyzed by Glu159 and an inter-molecular interaction catalyzed by Ntn, have been proposed for the reaction [21, 25, 28]
Summary
Plasmids, and Chemicals—Glutaric anhydride, succinate anhydride, glutaric acid, glutathione (GSH), 6-nitro-3phenylacetamidobenzoic acid, and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai) and Aldrich. Site-directed Mutagenesis—For mutagenesis of the CA spacer peptide region, a BsaI site was created upstream of the internal BamHI using PCR primers 5Ј-CAGCCATCATCATCATCATCAC-3Ј and 5Ј-TTGGATCCTTGATCGGCCAGGTCCGGCGGGTCTC-3Ј, and a SmaI site was introduced downstream of the natural internal BamHI site using primers, 5Ј-AAGGATCCAACTCCTGGGCGGTGGCCCCGGGAA-3Ј and 5Ј-CACTATAGGGCGAATTGGGT-3Ј. Introduction of these two restriction sites did not change the CA aa sequence. To identify the spacer released from CA mutants (E159D, E159Q, S170C, and m15), the purified enzyme was incubated in PBS buffer (pH 8) for 10 h at 30 °C. The gradient for long peptide separation was 5–95% acetonitrile in 0.1% formic acid over 30 min
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