Detection of covalent enzyme-substrate complexes of nitrilase by ion-spray mass spectroscopy.
Nitrilase from Rhodococcus ATCC 39484 was found to consist of two species of Mr 40,258 +/- 2 and 40,388 +/- 2 Da. When the enzyme was incubated with nitrile substrates and the reaction quenched with acid, higher Mr species were observed. The mass differences were consistent with addition of a substrate molecule to each species. These results represent the first reported demonstration that this, or any other nitrilase forms a covalent intermediate with its substrates. The observation that the intermediate, suggested to be either a thioimidate or an acylenzyme, can be trapped by acidification indicates that the rate of breakdown of the intermediate is rate-limiting.
Stevenson
D E
DE
National Research Council of Canada, Biotechnology Research Institute, Montreal.
Feng
R
R
Storer
A C
AC
D000619:Aminohydrolases / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D009570:Nitriles / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D012240:Rhodococcus / Q000201:enzymology
D009570:Nitriles; D000619:Aminohydrolases; C021867:nitrilase