Alteration of the specificity of ecotin, an E. coli serine proteinase inhibitor, by site directed mutagenesis.
The gene of ecotin, an E. coli proteinase inhibitor, was cloned, and by site-directed mutagenesis the active site residue of the protein, Met84, was mutated to Lys, Arg and Leu. The recombinant wild-type and mutant inhibitors were overexpressed in E. coli, purified to homogeneity and their inhibitory effects on trypsin, chymotrypsin and elastase were compared. Of these serine proteinases trypsin is the most strongly inhibited by wild type ecotin and its mutants. According to our results the character of residue 84 of ecotin significantly but not dramatically modifies the specificity of the inhibitor.
Pál
G
G
Department of Biochemistry, Eötvös University, Budapest, Hungary.
Sprengel
G
G
Patthy
A
A
Gráf
L
L
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins; D007930:Leucine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D033903:Periplasmic Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D014361:Trypsin Inhibitors
D001426:Bacterial Proteins; C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D001120:Arginine; D008715:Methionine; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D007930:Leucine; D008239:Lysine