Effects of site-directed mutations on processing and activities of penicillin G acylase from Escherichia coli ATCC 11105.
Penicillin G acylase from Escherichia coli ATCC 11105 is synthesized from its precursor polypeptide into a catalytically active heterodimer via a complex posttranslational processing pathway. Substitutions in the pair of aminoacyl residues at the cleavage site for processing the small and large subunits were made. Their processing phenotypes and penicillin G acylase activities were analyzed. By the introduction of a prolyl residue at either position, the processing of the small subunit was blocked without a change in enzymatic activity. Four other substitutions had no effect. At the site for processing the large subunit, four substitutions out of the seven examined blocked processing. In general, penicillin G acylase activity seemed to be proportional to the efficiency of the large-subunit-processing step. Ser-290 is an amino acid critical for processing and also for the enzymatic activity of penicillin G acylase. In the mutant pAATC, in which Ser-290 is mutated to Cys, the precursor is processed, but there is no detectable enzymatic activity. This suggests that there is a difference in the structural requirements for the processing pathway and for enzymatic activity. Recombination analysis of several mutants demonstrated that the small subunit can be processed only when the large subunit is processed first. Some site-directed mutants from which signal peptides were removed showed partial processing phenotypes and reduced enzymatic activities. Their expression showed that the prerequisite for penicillin G acylase activity is the efficient processing of the large subunit and that the maturation of the small subunit does not affect the enzymatic activity.
Choi
K S
KS
Department of Microbiology, College of Natural Sciences, Seoul National University, Korea.
Kim
J A
JA
Kang
H S
HS
D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D010399:Penicillin Amidase / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics*; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D021382:Protein Sorting Signals; D011392:Proline; D010399:Penicillin Amidase