Tyrosine 387 and arginine 404 are critical in the hydrolytic mechanism of Escherichia coli aminopeptidase P.
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.
Jao
Shu-Chuan
SC
Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan.
Huang
Li-Fang
LF
Hwang
Shu-Mei
SM
Li
Wen-Shan
WS
D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D006860:Hydrogen Bonding; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D014443:Tyrosine; D001120:Arginine; D000626:Aminopeptidases