PMID:10692453
| Citation |
Klenotic, PA, Carlos, JL, Samuelson, JC, Schuenemann, TA, Tschantz, WR, Paetzel, M, Strynadka, NC and Dalbey, RE (2000) The role of the conserved box E residues in the active site of the Escherichia coli type I signal peptidase. J. Biol. Chem. 275:6490-8 |
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| Abstract |
Type I signal peptidases are integral membrane proteins that function to remove signal peptides from secreted and membrane proteins. These enzymes carry out catalysis using a serine/lysine dyad instead of the prototypical serine/histidine/aspartic acid triad found in most serine proteases. Site-directed scanning mutagenesis was used to obtain a qualitative assessment of which 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 between 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 conserved salt bridge partners Asp-280 and Arg-282. Finally, Gly-272 is essential for 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-272 with a methyl group results in steric crowding, perturbation of the active site conformation, and specifically, disruption of the Ser-90/Lys-145 hydrogen bond. A refined model is proposed for the catalytic dyad mechanism of signal peptidase I in which the general base Lys-145 is positioned by Ser-278, which in turn is held in place by Asp-280. |
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| Keywords |
Bacterial Outer Membrane Proteins/metabolism; Bacterial Proteins/chemistry; Binding Sites; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Hydrogen Bonding; Membrane Proteins; Micrococcal Nuclease/metabolism; Models, Molecular; Mutagenesis; Mutation; Protein Conformation; Protein Precursors/metabolism; Serine Endopeptidases/chemistry; Serine Endopeptidases/genetics |
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