Kinetic constants of signal peptidase I using cytochrome b5 as a precursor substrate.
A procedure is described for measuring Escherichia coli signal peptidase I activity which exploits an intact precursor protein composed of the alkaline phosphatase signal peptide fused to the full length mammalian cytochrome b5. This cytochrome b5 precursor protein has been extensively characterised and shown to be processed accurately by purified signal peptidase I [Protein Expr. Purif. 7 (1996) 237]. The amphipathic, chimaeric cytochrome b5 precursor was isolated in mg quantities in a highly homogeneous state under non-denaturing conditions. The processing of the cytochrome b5 precursor by signal peptidase displayed Michaelis-Menten kinetics with K(m)=50 microM and k(cat)=11 s(-1). The K(m) was 20-fold lower than that obtained with signal peptide substrates and 3-fold higher than that reported for pro-OmpA-nuclease A precursor fusion. The corresponding turnover number, k(cat), was four orders of magnitude greater than the peptide substrates but was 2-fold lower than pro-OmpA-nuclease A precursor fusion. These results confirm that both the affinities and the catalytic power of the signal peptidase are significantly higher for macromolecular precursor substrates than for the shorter signal peptide substrates.
Gallagher
J
J
Institute of Biological Sciences, The University of Wales Aberystwyth, SY23 3DD, Aberystwyth, UK.
Kaderbhai
N N
NN
Kaderbhai
M A
MA
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D015786:Cytochromes b5; D000469:Alkaline Phosphatase; D012697:Serine Endopeptidases; C026515:type I signal peptidase