Nitrilase reaction mechanism

Mahadevan and Thimann [43] postulated the first nitrilase reaction mechanism, suggesting that the nitrile carbon present in the substrate displays a partial positive charge that is subjed to nudeophihc attack by one of the two SH groups in the nitrilase active site. The resulting thioimidate is then hydrolyzed to a thioester, with the release of ammonia as a by-product Hydrolysis of the acyl-enzyme then results in the release of the final acid product. 

Nitrilases and nitrile hydratases are distinct enzymes, apparently differing both with respect to prosthetic groups and reaction mechanisms. 

Nitrilases have been studied less than the nitrile hydratases. The enzymes appear as homomultimers, exhibiting a wide range of molecular masses. The reaction mechanism depicted in Fig. 12.1-2 has been proposed recently by Kobayashi et al. 2S. Several nitrilases have been found to be inhibited by reagents which bind to thiol groups, indicating that sulfhydryl groups are essential for the catalytic activity of... 

Figure 72.1-2. Reaction mechanism proposed for nitrilase catalysis1281.

Nitrilases are classified into branch 1 of the nitrilase superfamily, which is comprised of enzymes acting on various nonpeptide CN bonds [15]. All the proteins of this superfamily are characterized by a conserved catalytic triade (glu, lys, cys) and an additional conserved glu residue that seems to participate in the reaction mechanism [2]. Members of class 1 transform the CN bonds in nitriles and cyanides. The enzymes in which these activities were confirmed share in some cases levels of aa sequence identity as low as about 20%. This sequence diversity is reflected in different substrate specificities and different reaction products (carboxylic acids, amides) in various subtypes of these enzymes (aromatic nitrilases, aliphatic nitrilases, arylacetonitrilases, cyanide hydratases, cyanide dihydratases). 

There are many examples of nitrilase-catalyzed reactions in which amides form a considerable amount of the reaction products, such as the transformations of acrylonitrile analogs and a-fluoroarylacetonitriles by nitrilase 1 from Arabidopsis thaliana [17], the conversion of p-cyano-L-alanine into a mixture of L-asparagine and L-aspartic acid by nitrilase 4 from the same organism [18] or the transformations of mandelonitrile by nitrilase from Pseudomonas jhiorescens [19] or some fungi [8], Moreover, formamide is the only product of the cyanide transformation by cyanide hydratase. Therefore, this enzyme was classified as a lyase (EC 4.2.1.66), although it is closely related to nitrilases, as far as its aa sequence and reaction mechanism are concerned [3]. 

Aliphatic amidase enzymes demonstrate sequence similarity to the nitrilase superfamily thus indicating some form of evolutionary relationship. These amidases contain a Glu-Lys-Cys catalytic triad and exist as homotetrameric or homohexameric sttuctures that function via a ping-pong (bi-bi) reaction mechanism [60, 61]. 

Generally accepted mechanism for nitrilase-catalyzed hydration of organic nitriles to carhoxylic acids (bottom right, 16) and possible side reaction leading to amides (top right, 14) Cys-SH active site (qrsteine residue [21,22]. 

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