Identification and localization of two membrane-bound esterases from Escherichia coli

Abstract

Hydrolytic activities of isolated membrane fractions of Escherichia coli against chromogenic substrates, p-nitrophenyl ester and ,-naphthyl ester derivatives of N-substituted amino acids, were investigated by spectrophotometric and electrophoretic methods. Although detergents were absolutely necessary for the solubilization of enzymes, the amount of solubilized activities was increased by adding salt, such as NaCl or KCl. Two esterases were identified and separated by PAGE and by chromatography of the solubilized proteins in the presence of detergent. One hydrolyzed the alanine derivatives preferentially, whereas the other was mainly active on phenylalanine derivatives. Only the first was inactivated by diisopropyl fluorophosphate, a serine hydrolase inhibitor. Whereas the chymotrypsin-like enzyme was equally distributed between the inner and the outer membrane, the alanine activity was only detected in the inner membrane. They were both resistant to extraction with high salt concentrations, indicating their integral association with membranes. A study of the accessibility of these enzymes to their substrate in membrane vesicles with known polarity suggests that both alanine and phenylalanine activities are localized near the external surface of the cytoplasmic (inner) membrane. However, the phenylalanine activity (chymotrypsin-like enzyme) appears to be deeply buried inside the outer membrane. Because of its insensitivity to diisopropyl fluorophosphate, this last esterase seems to be distinct from the previously isolated periplasmic endopeptidase, protease I, which is also a chymotrypsin-like enzyme. Several lines of evidence indicate that membrane-bound proteolytic enzymes may play an important role in bacterial and eucaryotic cell physiology. The occurrence of limited proteolysis in bacteria has been reported in processes such as the transfer of secreted proteins (reviewed in references 5, 7, 26), membrane biogenesis (10, 12), and penetration of toxins into sensitive cells (22). However, the mechanism and physiological significance of these specific cleavages are still obscure. It is, therefore, of interest to undertake a detailed study of the enzymes involved in these reactions. All endopeptidases so far purified and characterized in Escherichia coli are soluble proteins. One of them, protease I (19, 20), is localized in the periplasmic space, whereas the other two, protease II (17, 18) and protease III (3) are found in the cytoplasm. Very little is known, however, about endopeptidases existing in membranes. Attempts to characterize particulate enzymes have been hampered by the lack of suitable solubilization procedures, instability of the solu

Purification and characterization of a heat-stable esterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

“…Several esterases from mammalian sources have been purified and studied in detail (Krisch, 1971). Ester-cleaving enzymes from other eukaryotic sources and from eubacteria have also been described, including the esterases from Aspergillus (Iwai et al, 1983) and bacteria such as Escherichia coli (Pacaud, 1982), Pseudomonas species (Shum & Markovetz, 1974a,b;Ohkawa et al, 1979), Mycobacterium (Akao et al, 1981) and members of the family of Bacillaceae (Higerd & Spizizen, 1973;Matsunaga et al, 1974). We found an esterase activity in Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium, which grows at 70°C and pH 2.…”

Molecular cloning of extremely thermostable esterase gene from hyperthermophilic archaeonPyrococcus furiosus inEscherichia coli

“…Microbial esterases in particular are desirable candidates for industrial use compared to relatively expensive esterases from mammalian sources. A large number of microbial esterases have been described, including esterases from Escherichia (Pacaud, 1982), Bacillus (Higerd and Spizizen, 1973;Matsunaga et al, 1974;Wood et al, 1995), Pseudomonas (Nakagawa et al, 1984;Ohkawa et al, 1979;Shum and Markovetz, 1974), Thermoanaerobacterium (Shao and Wiegel, 1995), Caldocellum (Luthi et al, 1990), Aspergillus (Okumura et al, 1983), and Sulfolobus species (Sobek and Gorisch, 1988). Some of these enzymes are relatively thermostable esterases from moderate or extreme thermophiles that grow at temperatures from 60 to 85°C (Luthi et al, 1990;Matsunaga et al, 1974;Shao and Wiegel, 1995;Sobek and Gorisch, 1988;Wood et al, 1995).…”

Detection of several diisopropylfluorophosphate-binding proteins in the outer membrane of Escherichia coli K-12

“…wide variety of proteins can be substrates. These substrates include colicins [2][3][4][5], ferricenterochelin receptor [6], M13 precoat protein [7], alkaline phosphatase precursor protein [8], nitrate reductase [9], casein [10], plasminogen [11], and also synthetic protease substrates [12]. Only a few of the proteases have been purified and characterized [7,13,14].…”