Kinetic and mechanistic studies of signal peptidase I from Escherichia coli

Signal peptidases of prokaryotic organisms reside in the outer leaflet of t he cytoplasmic membrane and catalyze the hydrolytic cleavage of a specific peptide bond of membrane-imbedded preproteins to liberate mature proteins f or secretion. In this manuscript, we report new and efficient peptide subst rates for SPase and their use to explore features of this enzyme's reaction mechanism. The enzyme used in this study was recombinant SPase I of Escher ichia coli that had been solubilized with Triton X-100 and purified to near homogeneity. Our new substrates are based on the fluorogenic peptide repor ted by Zhong and Benkovic [(1998) Anal. Biochem. 255, 66], Y(NO2)FSASALA si milar to KIKAbz-NH2 (Y-NO2, 3-nitro-L-tyrosine; K-Abz, epsilon-(2-aminobenz oyl)-L-lys; hydrolysis at A-K). We found that when a signal peptidelike seq uence is appended onto the N-terminus of this peptide to produce K-5-L(10)Y (NO2)FSASALA similar to KIKAbz- NH2, k(c)/K-m increases from 85 to 2.5 x 10 (6) M-1 s(-1). k(c)/K-m decreases with increasing concentration of Triton X -100 micelles under the condition [Triton X-100](micelle) > [S](0) > [E](0) . We explain this apparent inhibition with a model of surface dilution kine tics in which "empty" micelles compete with substrate-containing micelles f or micelle-bound enzyme. Fusion of micelle-bound enzyme with a substrate-co ntaining micelle leads to formation of productive E:S substrate complexes w hile fusion of micelle-bound enzyme with an "empty" micelle is nonproductiv e and inhibitory. The dependence of steady-state kinetic parameters for the SPase-catalyzed hydrolysis of K-5-L-10-Y(NO2)FSASALA similar to KIKAbz-NH2 on [Triton X-100](micelle) supports this model. Product inhibition and sol vent isotope effects were also investigated and could be interpreted in the context of this model.