Protease I from Escherichia coli

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Abstract
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‐tyrosine‐p‐nitrophenyl ester at pH 7.5 in 100 mM sodium phosphate buffer at 25 oC 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 oC, 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.