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.Penicillin G acylase (PGA) is an enzyme that catalyzes the hydrolysis of benzylpenicillin to give 6-aminopenicillanic acid, an intermediate in the production of semisynthetic penicillin (28). PGA from Escherichia coli ATCC 11105 is a periplasmic enzyme that is composed of the small subunit and the large subunit. For PGA to reach its catalytically active dimer form in the periplasm, multistep proteolytic processing from a single polypeptide must be accomplished by removing a signal peptide (26 residues) and a spacer peptide (54 residues) (24, 25). The proteolytic cleavage sites are located between the Ala-235-Ala-236 and Thr-289-Ser-290 pairs (18). This maturation mechanism is peculiar to PGA as a bacterial enzyme, though many viral proteins and eucaryotic polypeptide hormones require activation by proteolytic cleavage processes (6,7). But the enzymes involved in each processing step and their locations within the cell have not yet been identified.In this report, site-specific mutations at the proteolytic cleavage sites of PGA were constructed in order to obtain the necessary information for cleavage activation, especially (i) the amino acids critical to PGA processing and PGA activity, (ii) the amino acid specificity of each cleavage step, and (iii) the order of the cleavage steps. Through the expression of various recombinantpga genes, the characteristics of each...