Biogenesis and secretion of alkaline phosphatase and its mutants in Escherichia coli. III. Substitution of N-terminal amino acids of alkaline phosphatase affect its biogenesis

• Published in: Molekuliarnaia biologiia Vol. 28; no. 2; p. 374
• Main Authors: Karamyshev, A L, Kalinin, A E, Khmel'nitskiĭ, M I, Shliapnikov, M G, Ksenzenko, V N, Nesmeianova, M A
• Format: Journal Article
• Language: Russian
• Published: Russia (Federation) 01.03.1994
• Subjects: Alkaline Phosphatase - biosynthesis; Alkaline Phosphatase - genetics; Alkaline Phosphatase - metabolism; Amino Acid Sequence; Amino Acids - genetics; Base Sequence; Enzyme Precursors - metabolism; Escherichia coli - enzymology; Molecular Sequence Data; Mutation; Plasmids; Protein Processing, Post-Translational; RNA, Bacterial - genetics; RNA, Transfer - genetics
• ISSN: 0026-8984

The effect of the N-terminal amino acid substitution on E. coli alkaline phosphatase biogenesis has been studied. The substitutions of Ser, Gln, Tyr, Leu, Gly, Ala, Glu, Phe, His, Cys, Lys and Pro for Arg(+1) were obtained by creating amber mutation at the corresponding position within phoA gene and expressing this mutated gene in E. coli strains that produce the amber-suppressor tRNAs. All mutant proteins were shown to translocate across the cytoplasmic membrane and possess enzyme activity. The introduction of Pro in +1 position disturbs the cleavage of signal peptide whereas the insertion of the other amino acids does not change the rates of processing in comparison with wild-type protein. All amino acid substitutions affect alkaline phosphatase isoenzyme composition. Some experimental evidence were also obtained on the specificity of protease, which split off N-terminal Arg during alkaline phosphatase maturation.