IDENTIFICATION OF 3 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 Ach romobacter lyticus M497-1 extracellularly secretes. The structural asp ects necessary for the protease to function were investigated by means of site-directed mutagenesis to identify the constituents of the cata lytic triad and the amino acid residue responsible for lysine specific ity. The precursor molecules, which were produced by substitution of H is-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 autocata lytically and attained full protease activity. Substitution of Glu-190 , one of the two candidates for determining lysine specificity, to glu tamine, aspartic acid, or leucine had no or little effect on both prot eolytic activity and substrate specificity. However, the kinetic param eters were subtly different from one another, depending on the nature of substituents in these mutants. The substitution of the other candid ate, Asp-225, for asparagine or leucine resulted in the failure of mat uration to the active forms. However, the precursor of the D225E mutan t slowly matured and was essentially inactive. The observed reduction of protease activity is largely due to a decrease in the affinity of l ysine to the protease. These results suggest that His-57, Asp-113 and Ser-194 are the three constituents of the catalytic triad in Achromoba cter protease I and that Asp-225 plays a critical role in restricted s ubstrate specificity as a lysyl endopeptidase.