Mutational evidence of transition state stabilization by Serine 88 in Escherichia coli type I signal peptidase

Type I signal peptidase (SPase I) catalyzes the hydrolytic cleavage of the N-terminal signal peptide from translocated preproteins. SPase I belongs to a novel class of Ser proteases that utilize a Ser/Lys dyad catalytic mecha nism instead of the classical Scr/His/Asp triad found in most Ser proteases , Recent X-ray crystallographic studies indicate that the backbone amide ni trogen of the catalytic Ser 90 and the hydroxyl side chain of Ser 88 might participate as H-bond donors in the transition-state oxyanion hole. In this work, contribution of the side-chain Ser 88 in Escherichia coli SPase I to the stabilization of the transition state was investigated through in vivo and in vitro characterizations of Ala-, Cys-, and Thr-substituted mutants. The S88T mutant maintains near-wild-type activity with the substrate pro-O mpA nuclease A. In contrast, substitution with Cys at position 88 results i n more than a 740-fold reduction in activity (k(cat)) whereas S88A retains much less activity (>2440-fold decrease). Measurements of the kinetic const ants of the individual mutant enzymes indicate that these decreases in acti vity are attributed mainly to decreases in k(cat) while effects on K-m are minimal. Thermal inactivation and CD spectroscopic analyses indicate no glo bal conformational perturbations of the Ser 88 mutants relative to the wild -type E. coli SPase I enzyme. These results provide strong evidence fur the stabilization by Ser 88 of the oxyanion intermediate during catalysis by E . coli SPase I.