西亚试剂：The N-terminal nucleophile serine of cephalosporin acylase

The N-terminal nucleophile serine of cephalosporin acylase executes the second autoproteolytic cleavage and Acyl-peptide hydrolysis

Jun Yin, Zixin Deng, Guoping Zhao and Xi Huang

Cephalosporin acylase 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 E159 and G160, and detected the corresponding 10 aa spacer G160DPPDLADQG169 of CA mutants. The site of the second cleavage depended on E159: moving E into the spacer or removing 5-10 residues from the spacer sequence resulted in shorter spacers with the cleavage at the carboxylic side of E. The mutant E159D was cleaved more slowly than the wild-type CA, as were the two mutants G160A and G160L. This allowed kinetic measurements showing that the second cleavage reaction was a first-order, intra-molecular process. Glutaryl-7-amino-cephalosporanic acid (Gl-7-ACA) is the classic substrate of CA, in which the N-terminal S170 of the β-subunit, is the nucleophile. E and D resemble glutaryl, suggesting that CA might also remove N-terminal E or D from peptides. This was indeed the case, suggesting that the N-terminal nucleophile (Ntn) also performed the second proteolytic cleavage. We also found that CA is an acyl-peptide hydrolase rather than a previously expected acyl-amino 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 Ntn mechanism, during which large conformational changes in the α-subunit C-terminal region are required to bridge the gap between E159 and S170.