Mutations in the proteolytic domain of Escherichia coli protease Lon impair the ATPase activity of the enzyme.
Conserved residues of the proteolytic domain of Escherichia coli protease Lon, putative members of the classic catalytic triad (H665, H667, D676, and D743) were identified by comparison of amino acid sequences of Lon proteases. Mutant enzymes containing substitutions D676N, D743N, H665Y, and H667Y were obtained by site-directed mutagenesis. The mutant D743N retained the adenosine triphosphate (ATP)-dependent proteolytic activity, thereby indicating that D743 does not belong to the catalytic site. Simultaneously, the mutants D676N, H665Y, and H667Y lost the capacity for hydrolysis of protein substrates. The ATPase activity of these three mutants was decreased by more than an order of magnitude, which suggests a close spatial location of the ATPase and proteolytic active sites and their tight interaction in the process of protein degradation.
Starkova
N N
NN
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow. nr@enzyme.siobc.ras.ru
Koroleva
E P
EP
Rumsh
L D
LD
Ginodman
L M
LM
Rotanova
T V
TV
D049069:ATP-Dependent Proteases; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D049070:Protease La; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*
D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; C483567:Lon protein, E coli; D049070:Protease La; D000251:Adenosine Triphosphatases