ABSTRACT
Individual subunits of penicillin acylase from E. coli were isolated by gel-filtration under denaturing conditions (8 M urea). Recovery of the catalytic activity of the penicillin acylase heterodimer was studied after removal of urea. In the case of the heterodimer, 40-60% of the initial activity was recovered, whereas the activity of individual subunits was not recovered. Combination of native enzyme subunits with subunits isolated from the enzyme pre-inactivated with the irreversible inhibitor phenylmethylsulfonyl fluoride resulted in heterodimers which were active only in the case of involvement of the beta-subunit of the active enzyme.
Subject(s)
Enzyme Inhibitors/pharmacology , Escherichia coli/enzymology , Penicillin Amidase/metabolism , Urea/pharmacology , Dimerization , Enzyme Activation , Kinetics , Penicillin Amidase/chemistryABSTRACT
The behavior of a penicillin acylase from E. coli was studied in the reversed-micelle system AOT--H2O--octane. Kinetic studies of the enzymatic hydrolysis of the m-carboxy-p-nitroanilide of phenylacetic acid, titration of the penicillin acylase active site with an irreversible specific inhibitor (phenylmethylsulfonyl fluoride), sedimentation analysis at different hydration degrees, and chemical modification showed that the enzyme loses no more than 20% of its initial activity during 3-4 h in the reversed-micelle systems of different hydration degrees and retains its catalytically active structure.
Subject(s)
Escherichia coli/enzymology , Penicillin Amidase/metabolism , Catalysis , Dioctyl Sulfosuccinic Acid , Enzyme Inhibitors/pharmacology , Micelles , Octanes , Penicillin Amidase/antagonists & inhibitors , Phenylmethylsulfonyl Fluoride/pharmacology , WaterABSTRACT
The Database of Ribosomal Cross-links (DRC) was created in 1997. Here we describe new data incorporated into this database and several new features of the DRC. The DRC is freely available via World Wide Web at http://visitweb.com/database/ or http://www. mpimg-berlin-dahlem.mpg.de/ approximately ag_ribo/ag_brimacombe/drc/
Subject(s)
Databases, Factual , Escherichia coli/metabolism , RNA, Ribosomal/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cross-Linking Reagents , Information Storage and Retrieval , Internet , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Ribosomal/chemistry , Ribosomal Proteins/chemistry , Ribosomes/chemistryABSTRACT
Penicillin acylase substrates suitable for colorimetric determination of the enzyme activity have been tested in this study. The kinetic parameters (Km and kcat) have been elucidated for the following nine substrates: six phenylacetic acid derivatives (p-nitroanilide, p-nitrophenyl ester, p-nitro-m-carboxyanilide, p-nitro-o-carboxyanilide, p-nitro-o-hydroxyanilide, m-nitro-p-carboxyanilide), two D-phenylglycine derivatives (p-nitroanilide, p-nitro-m-carboxyanilide), and also p-nitrophenyl ester of acetic acid (p-nitrophenyl acetate). With the exception of p-nitrophenyl acetate, all the compounds studied are highly specific chromogenic substrates for penicillin acylase, but their reactivity is very variable and kcat/Km values are in a range from 0.8.10(4) to 5.10(6) M(-1).sec(-1).
