The Complete Amino Acid Sequence of the Zn2+‐Containing d‐Alanyl‐d‐Alanine‐Cleaving Carboxypeptidase of Streptomyces albus G

Publisher Website
Google Scholar
Abstract
The 22,076-MW Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of S. albus G effectively catalyzes the transfer of the N.alpha.,N.epsilon.-diacetyl-L-lysyl-D-alanyl electrophilic group of the standard tripeptide substrate N.alpha.,N.epsilon.-diacetyl-L-lysyl-D-alanyl-D-alanine to water. It also performs a weak .beta.-lactamase activity, hydrolyzing penicillin into penicilloate at a very low rate. This protein consists of 212 amino acid residues in a single polypeptide chain. The N terminus is partially blocked as a result of the cyclization of the dipeptide Asn-Gly into anhydroaspartylglycine imide. The protein was fragmented by cyanogen bromide into 5 fragments whose sequences were determined via appropriate subcleavages with various proteases. The ordering of the cyanogen bromide peptide fragments was carried out by submitting the S-carboxymethylated protein to complete tryptic digestion and labeling the methionine-containing peptides obtained with iodo[14C]-acetamide and by submitting to limited tryptic digestion on the S-[2-(4''-pyridyl)ethyl]-cysteine protein whose amino groups were blocked by reaction with exo-cis-3,6-endoxo-.DELTA.4-tetrahydrophthalic anhydride prior to digestion. The protein contains 6 cysteine residues in the form of 3 disulfide bridges. No homology is found by comparing this peptidase with other Zn2+-containing enzymes (carboxypeptidase A, thermolysin, carbonic anhydrase B and alcohol dehydrogenase) and several completely or partially sequenced, serine-containing D-alanyl-D-alanine-cleaving peptidases and Zn2+ serine-containing .beta.-lactamases.