The role of the conserved Box E residues in the active site of the Escherichia coli type I signal peptidase

Type I signal peptidases are integral membrane proteins that function to re move signal peptides from secreted and membrane proteins. These enzymes car ry out catalysis using a serine/lysine dyad instead of the prototypical ser ine/histidine/aspartic acid triad found in most serine proteases. Site-dire cted scanning mutagenesis was used to obtain a qualitative assessment of wh ich residues in the fifth conserved region, Box E, of the Escherichia coli signal peptidase I are critical for maintaining a functional enzyme, First, we find that there is no requirement for activity for a salt bridge betwee n the invariant Asp-273 and the Arg-146 residues. In addition, we show that the conserved Ser-278 is required for optimal activity as well as conserve d salt bridge partners Asp-280 and Arg-282. Finally, Gly-272 is essential f or signal peptidase I activity, consistent with it being located within van der Waals proximity to Ser-278 and general base Lys-145 side-chain atoms. We propose that replacement of the hydrogen side chain of Gly-212 with a me thyl group results in steric crowding perturbation of the active site confo rmation, and specifically, disruption of the Ser-90/Lys-145 hydrogen bond. A refined model is proposed for the catalytic dyad mechanism of signal pept idase I in which the general base Lys-145 is positioned by Ser-278, which i n turn is held in place by Asp-280.