The study of biogenesis and secretion of alkaline phosphatase and its mutant forms in Escherichia coli. II. Effect of amino acid substitutions in the processing site and in N-terminus of mature polypeptide chain of alkaline phosphatase on its biogenesis

A. L. Karamyshev, A. E. Kalinin, I. M. Tsfasman, V. N. Ksenzenko, M. A. Nesmeyanova

Research output: Contribution to journalArticle

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Abstract

The effect of amino acid substitutions in E. coli alkaline phosphatase on its biogenesis has been studied. The substitution of Val for Ala(-1) in the signal peptide cleavage site completely inhibits all stages of posttranslational modification: processing and formation of isozymes. The absence of processing does not prevent translocation of the precursor across the cytoplasmic membrane and formation of an active enzyme macromolecule. The precursor of the above mutant protein was found in the periplasm and in the cytoplasmic membrane. The substitution of Gln for Glu(+4), as well as the double substitution of Ala for Arg(+1) and Gln for Glu(+4), in the N-terminus of mature polypeptide chain result in the change in the isozyme spectrum. Differences in the rates of processing in vivo of both mutant proteins were not revealed. However, the double amino acid substitution significantly increases the efficiency of the vitro processing. All amino acid substitutions studied have no effect on the peculiarities of biogenesis which are conditioned by oversynthesis of the enzyme encoded by the phoA gene in the plasmid: secretion into the culture medium and accumulation of precursor as insoluble aggregates in the cytoplasm. However, extracellular activities of mutant proteins differ from that of the wild-type protein, which may result from the change either in the efficiency of their secretion or in their catalytic properties.

Original languageEnglish (US)
Pages (from-to)362-373
Number of pages12
JournalMolekulyarnaya Biologiya
Volume28
Issue number2
StatePublished - Jan 1 1994

ASJC Scopus subject areas

  • Medicine(all)

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