Protease I from Escherichia coli. Some physicochemical properties and substrate specificity.
Protease I, a periplasmic endopeptidase from Escherichia coli has been further purified by a modified procedure. While the purified protein consists of a single polypeptide chain of about 21000 daltons, its molecular weight in dilute salt solution was estimated to be near 43000, suggesting that the enzyme has a marked tendency to dimerize. It has only one disulphide bond and is very sensitive to urea. In agreement with previous evidence of a chymotrypsin-like specificity, hydrolytic assays of various p-nitrophenyl esters of N-substituted amino acids showed that phenylalanine and tyrosine derivatives are the best substrates for the enzyme. The Km(app) for N-benzoyloxycarbonyl-L-tyrosin-p-nitrophenyl ester at pH 7.5 In 100 mM sodium phosphate buffer at 25 degrees C was found to be 0.2 mM. In contrast to chymotrypsin, protease I is unable to hydrolyse N-acetyl-L-phenylalanine ethyl ester and its tyrosine analogue. Moreover, the enzyme appears devoid of amidase activity and exhibits a low activity upon polypeptides. At 37 degrees C, it cleaves the carboxymethylated B-chain of bovine insulin at four points: Phe25-Tyr26, Phe24-Phe25, Leu15-Tyr16 and Ser9-His10. From a detailed study of peptides bonds hydrolyzed, it was concluded that protease I has a stringent requirement for both residues forming the scissile bond, and appears to possess an extended hydrophobic binding site.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D004220:Disulfides / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology*; D007122:Immunoelectrophoresis; D007328:Insulin; D007700:Kinetics; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D013329:Structure-Activity Relationship; D014508:Urea
D000596:Amino Acids; D004220:Disulfides; D007328:Insulin; D014508:Urea; D010447:Peptide Hydrolases