Protease I from Escherichia coli

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 21 000 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 K, (app) for N-benzoyloxycarbonyl-L-tyrosinep-nitrophenyl ester at pH 7.5 in 100 mM sodium phosphate buffer at 25 "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 "C, it cleaves the carboxymethylated B-chain of bovine insulin at four points : Phe25-Tyr26, Phe24-Phe25, Leu"-Tyrt6 and Ser9-His". 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.

Link between allergic asthma and airway mucosal infection suggested by proteinase-secreting household fungi

“…Although our detecting method is not definitive, it is likely that this dust proteinase is aspergillopepsin I (aspergillopeptidase A; extracellular acid protease; proteinase B), which has been variously reported to exist as ∼22 and ∼43 kDa proteins. 28,31,32 Microbial proteinases are known to multimerize in solution and it is likely that our ∼86 kDa product represents a tetramer of aspergillopepsin I 33 (Figure 1; and P Kolattukudy personal communication). Aspergillopepsins are secreted by many aspergillus species and we have used the highly allergenic A. oryzae aspergillopepsin for many years to generate allergic lung disease in mice.…”

Immunochemical Quantitation of Pancreatic Endopeptidases in the Intestinal Contents of Germfree and Conventional Rats

“…One apparent hazard with such analyses is that enzymological methods may give unspecific reactions caused by bacterial enzymes with tryptic or chymotryptic activity. It has been postulated that intestinal bacteria possessed no such activity (1 3, 19), but proteases with tryptic and chymotryptic activity were recently isolated from E. coli (29,30).…”

The Study of Escherichia coli Proteases. Intracellular Serine Protease of E. coli - an Analogue of Bacillus Proteases

“…Only the activity which was tightly bound to gramicidin S-Sepharose 4B was studied further. Pacaud et al (1976) found that E. coli contained protease I, an enzyme capable of cleavingp-nitrophenyl esters of N-substituted amino acids and possessing a 'chymotrypsinlike' substrate specificity. It may well be that this enzyme could cleave Z-L-Ala-L-Ala-L-LeupNA and possibly the activity towards this substrate that did not bind to gramicidin SSepharose 4B was protease I.…”

“…The scarcity of biochemical data on the purified E. coli enzymes is caused by difficulties in their isolation, the extremely low concentration within the cells an'd the absence of specific affinity adsorbents suitable for the simultaneous isolation of several proteases. ' Chymotrypsin-like' protease I (Pacaud & Uriel, 1971 ;Pacaud et al, 1976) and 'trypsin-like' protease I1 (Pacaud & Richaud, 1975;Pacaud, 1976) are the only endopeptidases of E. coli to have been purified and studied in some detail.…”