Identification of three catalytic triad constituents and Asp-225 essential for function of lysine-specific serine protease, Achromobacter protease I.
Achromobacter protease I is a lysine-specific serine protease that Achromobacter lyticus M497-1 extracellularly secretes. The structural aspects necessary for the protease to function were investigated by means of site-directed mutagenesis to identify the constituents of the catalytic triad and the amino acid residue responsible for lysine specificity. The precursor molecules, which were produced by substitution of His-57, Asp-113, or Ser-194 for alanine, could not be converted to the mature form. In contrast, a precursor of a mutant in which either His-56 or Ser-193 is converted to alanine was perfectly processed autocatalytically and attained full protease activity. Substitution of Glu-190, one of the two candidates for determining lysine specificity, to glutamine, aspartic acid, or leucine had no or little effect on both proteolytic activity and substrate specificity. However, the kinetic parameters were subtly different from one another, depending on the nature of substituents in these mutants. The substitution of the other candidate, Asp-225, for asparagine or leucine resulted in the failure of maturation to the active forms. However, the precursor of the D225E mutant slowly matured and was essentially inactive. The observed reduction of protease activity is largely due to a decrease in the affinity of lysine to the protease. These results suggest that His-57, Asp-113, and Ser-194 are the three constituents of the catalytic triad in Achromobacter protease I and that Asp-225 plays a critical role in restricted substrate specificity as a lysyl endopeptidase.
Norioka
S
S
Division of Protein Chemistry, Osaka University, Japan.
Ohta
S
S
Ohara
T
T
Lim
S I
SI
Sakiyama
F
F
D000421:Alcaligenes / Q000201:enzymology*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D017931:DNA Primers / Q000737:chemistry; D007700:Kinetics; D008239:Lysine; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D012697:Serine Endopeptidases / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D017931:DNA Primers; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D008239:Lysine