STUDY OF BIOGENESIS AND SECRETION OF ALKALINE-PHOSPHATASE AND ITS MUTANT FORMS IN ESCHERICHIA-COLI .2. EFFECT OF AMINO-ACID SUBSTITUTIONS IN THE PROCESSING SITE AND THE N-TERMINUS OF MATURE POLYPEPTIDE-CHAIN ON ITS BIOGENESIS

Effect of amino acid substitutions in Escherichia coli alkaline phosph atase [EC 3.1.3.1] on its biogenesis was studied. The substitution of Val for Ala(-1) in the site of signal peptide cleavage blocks all step s of posttranslational modification: processing and formation of isozy mes. The lack of processing does not prevent the precursor translocati on across the cytoplasmic membrane and the formation of active enzyme. The precursor of this mutant protein was found in the periplasm and c ytoplasmic membrane. The substitution of Gin for Glu(+4), as well as d ouble substitution of Gin for Glu(+4) and Ala for Arg(+1) in the N-ter minal domain of alkaline phosphatase mature polypeptide chain result i n the change of its isozyme spectrum. No differences were revealed in the rate of in vivo processing of these mutant proteins. Double amino acid substitutions significantly increase the efficiency of in vitro p rocessing. Ah amino acid substitutions studied have no effect on aberr ant biogenesis resulting from enzyme overproduction by the plasmid-enc oded PhoA gene: its secretion into culture medium and accumulation of precursor as insoluble aggregates in the cytoplasm. However, the extra cellular activities of mutant proteins differ from that of the wild ty pe protein owing to the changes in their secretion efficiency or catal ytic properties.