Reconstitution of human azurocidin catalytic triad and proteolytic activity by site-directed mutagenesis.
Azurocidin belongs to the serprocidin family, but it is devoid of proteolytic activity due to a substitution of His and Ser residues in the catalytic triad. The aim of this study was to reconstitute the active site of azurocidin by site-directed mutagenesis, analyze its processing and restored proteolytic activity. Azurocidin expressed in Sf9 insect cells possessing the reconstituted His41-Asp89-Ser175 triad exhibited significant proteolytic activity toward casein with a pH optimum of approximately 8-9, but a reconstitution of only one active site amino acid did not result in proteolytically active protein. Enzymatically active recombinant azurocidin caused cleavage of the C-terminal fusion tag with the primary cleavage site after lysine at Lys-Leu and after alanine at Ala-Ala, and the secondary cleavage site after arginine at Arg-Gln, as well as with low efficiency caused cleavage of insulin chain B after leucine at Leu-Tyr and Leu-Cys, and after alanine at Ala-Leu. We demonstrate that cleavage of the azurocidin C-terminal tripeptide is not necessary for its enzymatic activity. The first isoleucine present in mature azurocidin can be replaced by similar amino acids, such as leucine or valine, but its substitution by histidine or arginine decreases proteolytic activity.
Olczak
Mariusz
M
Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Tamka 2, Wroclaw, Poland. Mariusz.Olczak@biotech.uni.wroc.pl
Indyk
Katarzyna
K
Olczak
Teresa
T
D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
C062991:AZU1 protein, human; D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D002352:Carrier Proteins; D011994:Recombinant Proteins; D010447:Peptide Hydrolases