Detection of covalent enzyme‐substrate complexes of nitrilase by ion‐spray mass spectroscopy

FEBS Letters

Storer, Andrew C.¹  Feng, Rong¹  Stevenson, David E.¹ 

[1] National Research Council of Canada, Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, H4P 2R2, Canada

关键词: Nitrilase;    Mass spectroscopy;    Nitrile hydrolysis;    Covalent catalysis;   

DOI  :  10.1016/0014-5793(90)80821-Y

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Nitrilase from Rhodococcus ATCC 39484 was found to consist of two species of M _r 40 258 ±2 and 40 388 ±2 Da. When the enzyme was incubated with nitrile substrates and the reaction quenched with acid, higher M _r, 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.

【 授权许可】

Unknown