Characterization of a Potential Catalytic Residue, Asp-133, in the High Affinity ATP-binding Site of Escherichia coli SecA, Translocation ATPase

Ken Sato,Hiroyuki Mori,Masasuke Yoshida,Shoji Mizushima

doi:10.1074/jbc.271.29.17439

Abstract

The high affinity ATP-binding site of SecA is located in its amino-terminal domain possessing amino acid sequences, the Walker A (GXXXXGKT) and B (ZZZZD) motifs, that are characteristic of a major class of nucleotide-binding sites (Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951). Recently, we proposed that proteins possessing a typical set of Walker A and B motifs contain a conserved Glu or Asp between the two motifs. This Glu or Asp acts as a "catalytic residue" that activates a water molecule for an in-line attack on the gamma-phosphate of ATP (Amano, T., Yoshida, M., Matsuo, Y., and Nishikawa, K.(1995) FEBS Lett. 359, 1-5). In the present study, the aspartate residue at position 133 in Escherichia coli SecA, which could be the "catalytic residue," was mutated to an asparagine. The mutant SecA (SecA D133N) protein was expressed in E. coli CK4706, encoding a duplication of the secA gene, and purified to homogeneity. The in vitro protein translocation activity and membrane vesicle stimulated ATPase activity of SecA D133N were drastically reduced. Proteolytic studies indicated that the conformational changes of the mutant SecA occurring on interaction with ATP, presecretory proteins, phospholipids, and membrane vesicles, were similar to those of wild-type SecA. The mutant SecA allowed the signal peptide cleavage of proOmpA during translocation, indicating that the mutant retains the ability to bind ATP to perform the initial step of the translocation reaction. These data indicate that the carboxyl group of Asp-133 plays a role as a catalytic carboxylate, which activates a water molecule to attack gamma-phosphate of ATP, and the mutant lacking this residue cannot perform the total translocation but can still perform the initial step of the protein translocation.

Highlights

The amino terminus of SecA contains typical ATP-binding motifs, the so-called Walker A and B sequences [11], that are commonly found in many nucleotide-binding proteins
Construction and Expression of the Mutant secA Gene—As shown in Fig. 1, when the amino acid sequences of the region covering Walker A and B motifs were aligned for several SecA proteins, it became clear that the occurrence of Glu or Asp at a position 24 Ϯ 2 residues away from the Lys of motif A is a common feature
To reveal the functions of these carboxyl groups and ATP hydrolysis in E. coli, Asp-133 of E. coli SecA was mutated to the cognate amide residue, asparagine

Summary

Introduction

The amino terminus of SecA contains typical ATP-binding motifs, the so-called Walker A and B sequences [11], that are commonly found in many nucleotide-binding proteins. Proteolytic studies indicated that the conformational changes of the mutant SecA occurring on interaction with ATP, presecretory proteins, phospholipids, and membrane vesicles, were similar to those of wild-type SecA. The endogenous, membrane, and translocation ATPase activities of the purified wild-type and mutant SecA were assayed (Fig. 2).

Results

Conclusion