Tyrosine 387 and Arginine 404 Are Critical in the Hydrolytic Mechanism of Escherichia coli Aminopeptidase P

Abstract

Analysis of the pH-rate profile for catalysis of bradykinin cleavage by aminopeptidase P (AMPP), a manganese-containing hydrolase from Escherichia coli, was carried out to show that optimal catalytic function is obtained at neutral pH. On the basis of information derived from the crystal structure, peptidase sequence alignments, and the hydrolysis of organophosphate triesters, active site residues Arg153, Arg370, Trp88, Tyr387, and Arg404 were identified as potential catalytic residues. Site-directed mutagenesis was used to substitute these residues with Leu, Ala, Trp, Lys, or Phe. The kcat values for the Arg153, Arg370, and Trp88 mutants were nearly the same as that for the wild-type enzyme. The kcat values of the R404K, R404A, and Y387A mutants were lower by factors of 285, 400, and 16, respectively. Inductively coupled plasma mass spectrometry and circular dichroism spectroscopy showed that Arg404 is not required for metal chelation or stabilization of protein secondary structure. The hydrogen bond network observed between the side chains of conserved residues Asp260, Arg404, and Tyr387 indicated that Arg404 participates in proton relay. This was further evidenced by the return of activity in the R404A mutant by the addition of guanidine. Also, reduced catalytic efficiency in the R404K mutant, which conserves the positive charge at the bridge site, shows that only the arginine group of Arg404 (not the ammonium group of Lys404) can participate in the hydrogen bond network. The hydrogen bond interaction between the Arg404 and the Tyr387 ring hydroxyl group is suggested by the reduced catalytic efficiency of the Y387F mutant.

Evaluation of Organophosphorus Chemicals‐Degrading Enzymes: A Comparison of Escherichia coli and Human Cytosolic Aminopeptidase P

“…The expression and purification of His-tagged E. coli AMPP and its mutants have been described in [4] [13]. The proteins were purified from BL21(DE3) cells according to the methods therein.…”

Crystallization and preliminary X-ray diffraction analysis of Xaa-Pro dipeptidase fromXanthomonas campestris

“…1). In aminopeptidase P from E. coli Tyr387 has been proposed to be involved in the conserved hydrogen-bond network in the Asp260-Arg404-Tyr387 motif, which is able to shuttle a proton from the bulk solvent to the leaving peptide (Jao et al, 2006). Moreover, it was also suggested that Tyr387, Arg404 and His350 are important residues for proline specificity (Wilce et al, 1998;Graham et al, 2006).…”

“…Moreover, it was also suggested that Tyr387, Arg404 and His350 are important residues for proline specificity (Wilce et al, 1998;Graham et al, 2006). Site-directed mutagenesis of Tyr387 and Arg404 resulted in a severalfold reduction in the activity of aminopeptidase P from E. coli (Jao et al, 2006). From this viewpioint, structural and functional characterization of XPD43 from X. campestris is necessary.…”

Proline-Specific Extracellular Aminopeptidase Purified from Streptomyces lavendulae

“…Proline aminopeptidase (PAP) is therefore required to remove the proline residues and is essential for the degradation of proline-rich peptides and proteins, such as collagen and gelatin [6]. The crystal structure and catalytic mechanism of the proline aminopeptidase from Escherichia coli has been reported [7,8]. The genus Streptomyces was proved to be a good source of various substrate-specific aminopeptidases such as leucine or methionine aminopeptidase [9,10].…”