Nitrilase of Rhodococcus rhodochrous J1. Conversion into the active form by subunit association.
Nitrilase-containing resting cells of Rhodococcus rhodochrous J1 converted acrylonitrile and benzonitrile to the corresponding acids, but the purified nitrilase hydrolyzed only benzonitrile, and not acrylonitrile. The activity of the purified enzyme towards acrylonitrile was recovered by preincubation with 10 mM benzonitrile, but not by preincubation with aliphatic nitriles such as acrylonitrile. It was shown by light-scattering experiments, that preincubation with benzonitrile led to the assembly of the inactive, purified and homodimeric 80-kDa enzyme to its active 410-kDa aggregate, which was proposed to be a decamer. Furthermore, the association concomitant with the activation was reached after dialysis of the enzyme against various salts and organic solvents, with the highest recovery reached at 10% saturated ammonium sulfate and 50% (v/v) glycerol, and by preincubation at increased temperatures or enzyme concentrations.
Nagasawa
T
T
Department of Biomolecular Science, Gifu University, Gifu, Japan. tonagasa@apchem,gifu-u.ac.jp
Wieser
M
M
Nakamura
T
T
Iwahara
H
H
Yoshida
T
T
Gekko
K
K
D000181:Acrylonitrile / Q000378:metabolism; D000619:Aminohydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000645:Ammonium Sulfate; D002209:Caprolactam / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D005990:Glycerol; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D009570:Nitriles / Q000378:metabolism / Q000494:pharmacology; D010420:Pentanes / Q000494:pharmacology; D020836:Protein Structure, Quaternary / Q000187:drug effects; D012240:Rhodococcus / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics
D009570:Nitriles; D010420:Pentanes; D002209:Caprolactam; C014356:benzonitrile; D000619:Aminohydrolases; C021867:nitrilase; D000181:Acrylonitrile; C403810:isovaleronitrile; D005990:Glycerol; D000645:Ammonium Sulfate