Assuntos
Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Protease La , Regulon , Proteínas Repressoras/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transativadores/genética , Vibrio/genética , Proteases Dependentes de ATP , Escherichia coli/enzimologia , Hidrólise , Mutação , PlasmídeosRESUMO
A new efficient substrate, Suc-Phe-Leu-Phe-SBzl, was proposed for studying the function of the Escherichia coli ATP-dependent Lon protease and its modified forms. The kinetic parameters of hydrolysis of the substrate were determined. The esterase activity of protease Lon was found to be nucleotide-regulated.
Assuntos
Trifosfato de Adenosina/química , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Proteínas de Choque Térmico/química , Oligopeptídeos/química , Protease La , Serina Endopeptidases/química , Proteases Dependentes de ATP , Proteínas de Choque Térmico/genética , Hidrólise , Cinética , Mutação , Serina Endopeptidases/genética , Especificidade por SubstratoRESUMO
Selective protein degradation is an energy-dependent process performed by high-molecular-weight proteases. The activity of proteolytic components of these enzymes is coupled to the ATPase activity of their regulatory subunits or domains. Here, we obtained the proteolytic domain of Escherichia coli protease Lon by cloning the corresponding fragment of the lon gene in pGEX-KG, expression of the hybrid protein, and isolation of the proteolytic domain after hydrolysis of the hybrid protein with thrombin. The isolated proteolytic domain exhibited almost no activity toward protein substrates (casein) but hydrolyzed peptide substrates (melittin), thereby confirming the importance of the ATPase component for protein hydrolysis. Protease Lon and its proteolytic domain differed in the efficiency and specificity of melittin hydrolysis.
Assuntos
Endopeptidases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Protease La , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Proteases Dependentes de ATP , Sequência de Aminoácidos , Catálise , Escherichia coli/química , Proteínas de Choque Térmico/isolamento & purificação , Hidrólise , Meliteno/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Serina Endopeptidases/isolamento & purificaçãoRESUMO
The functional domain boundaries of the ATP-dependent Lon proteases were identified by comparative analysis of the amino acid sequences of the enzymes from evolutionarily distant organisms. Modified forms of the Escherichia coli Lon protease with the elongated or substituted N-terminal domain and a truncated enzyme lacking the N-terminal domain were obtained through genetic engineering methods. Analysis of the enzymatic properties of the resulting modified forms of Lon protease revealed the importance of the N-terminal domain in its function.
