Mechanism nitrilase

It has been noted already that reports can be found in the literature, as far back as the 1960s, of modest amounts of amides being formed in the presence of nitrilases [20-22]. This side activity (see Figure 16.7) could not be accounted for by the commonly accepted nitrilase mechanism [21] and was largely neglected until quite recently. It was then shown, for example, that nitrile hydration in the presence of recombrnantly expressed and purified nitrilases from Arahidopsis thali-ana was a major pathway with some nitriles, in particular electron-deficient ones [23-25]. 

Figure 16.10 Proposecd nitrilase mechanism for the formation of acid (a) and amide (b) [5],...

FIGURE 4.4 Mechanism of nitrilase, nitrile hydratase, amidase on PAN. 

Kielbasinski, P., Rachwalski, M., Mikolajczyk, M., et al. 2008. Nitrilase-catalysed hydrolysis of cyanomethyl p-tolyl sulfoxide Stereochemistry and mechanism. Tetrahedron Asymmetry, 19 562-7. 

Novo, C., Tata, R., Clemente, A., et al. 1995. Pseudomonas aeruginosa aliphatic amidase is related to the nitrilase/cyanide hydratase enzyme family and Cys is predicted to be the active site nucleophile of the catalytic mechanism. EEBS letters, 367 275-9. 

The hypothetical nitrilase catalytic mechanism based on previous schemes (for a review see [6]), which has been recently refined to bring more understanding to the formation of two possible end-products [39], is discussed in Chapter 16. According to a generally accepted hypothesis, the catalytic mechanism involves a nucleophilic attack on the cyano carbon by the sulfhydryl group of the conserved... 

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.

Aromatic, heterocyclic, and certain unsaturated aliphatic nitriles are often directly hydrolyzed to the corresponding acids without formation of the intermediate free amide by a so-called nitrilase enzyme. The nitrile hydratase and nitrilase enzyme use distinctively different mechanisms of action. 

On the other hand, nitrilases operate by a completely different mechanism (Scheme 2.101). They possess neither coordinated metal atoms, nor cofactors, but act through an essential nucleophilic sulfhydryl residue of a cysteine [641, 642], which is encoded in the nitrilase-sequence motif Glu-Lys-Cys [643]. The mechanism of nitrilases is similar to general base-catalyzed nitrile hydrolysis Nucleophihc attack by the sulfhydryl residue on the nitrile carbon atom forms an enzyme-bound thioimidate intermediate, which is hydrated to give a tetrahedral intermediate. After the elimination of ammonia, an acyl-enzyme intermediate is formed, which (like in serine hydrolases) is hydrolyzed to yield a carboxyhc acid [644]. 

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). 

Figure 12.2 Hypothetical mechanism of the nitrilase-catalyzed transformation of nitriles into carboxylic acids or amides. (According to [3].) E-SH enzyme with a cysteine as catalytic nucleophile.

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]. 

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. 

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]. 

There may also be other ways in which enzyme complexes contribute to the evolution of novel or enhanced enzyme function. One mechanism is suggested by recent examples fiom monocots in which multimers with altered substrate binding affinities or specificities have arisen through the interaction of closely-related isoforms. In the case of nitrilase in Sorghum bicolor, individual isoforms of NIT4 are catalytically inactive, but... 

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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