[Isolation and certain properties of mutant alkaline phosphatase of Escherichia coli].
Natural and mutant alkaline phosphatases with amino acid substitutions in the processing site and N-terminal domain of the mature polypeptide chain Val for Ala(-1), Gln for Glu (+4) and simultaneously Gln for Glu (+4) and Ala for Arg (+1) have been isolated from the periplasm and cultural fluid of E. coli. It has been found that these substitutions have little effect on the dependence of the enzyme activity on pH, ionic strength and temperature but influence its isoenzymic spectrum and decrease (almost twofold) the maximal rate of the enzyme-catalyzed reaction. Extracellular enzymes display, in contrast with periplasmic ones, other catalytic properties (Vmax) and binding activity (Km). After translocation through the outer membrane all the enzymes display decreased Vmax and increased Km. These changes are especially well-pronounced in case of the mutant protein PhoA46 which contains an uncleaved signal peptide due to the impossibility of processing resulting from the substitution of Val for Ala(-1). The Vmax for this protein is decreased 20 times, while the Km is increased 4-fold. The protein also shows a higher (in comparison with other proteins) sensitivity towards proteolytic enzymes and is less resistant upon storage. The experimental data suggest that the changes in the N-end of alkaline phosphatase located at a long distance from its active center influence the enzyme function.
Nesmeianova
M A
MA
Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Moscow.
Krupianko
V I
VI
Kalinin
A E
AE
Kadyrova
L Iu
LIu
D000469:Alkaline Phosphatase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007527:Isoenzymes / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009994:Osmolar Concentration; D013696:Temperature
D007527:Isoenzymes; D000469:Alkaline Phosphatase
Vydelenie i nekotorye svoĭstva mutantnykh shchelochnykh fosfataz Escherichia coli.