An Unsual Cys‐Glu‐Lys Catalytic Triad is Responsible for the Catalytic Mechanism of the Nitrilase Superfamily: A QM/MM Study on Nit2

Abstract

Nitrilase 2 (Nit2) is a representative member of the nitrilase superfamily that catalyzes the hydrolysis of α-ketosuccinamate into oxaloacetate. It has been associated with the metabolism of rapidly dividing cells like cancer cells. The catalytic mechanism of Nit2 employs a catalytic triad formed by Cys191, Glu81 and Lys150. The Cys191 and Glu81 play an active role during the catalytic process while the Lys150 is shown to play only a secondary role. The results demonstrate that the catalytic mechanism of Nit2 involves four steps. The nucleophilic attack of Cys191 to the α-ketosuccinamate, the formation of two tetrahedral enzyme adducts and the hydrolysis of a thioacylenzyme intermediate, from which results the formation of oxaloacetate and enzymatic turnover. The rate limiting step of the catalytic process is the formation of the first tetrahedral intermediate with a calculated activation free energy of 18.4 kcal/mol, which agrees very well with the experimental k cat (17.67 kcal/mol).

The Catalytic Mechanism of Pdx2 Glutaminase Driven by a Cys‐His‐Glu Triad: A Computational Study

“…The high reaction energy can be justified by the formation of two point charges in the residues Cys 87 and His 196 . The results show that this reaction is accomplished in a single step and, contrarily to what is proposed in the literature for other amidases (e. g. Nitrilase 2), [38] there is no formation of a second tetrahedral intermediate upon reaction with the water molecule.…”

The critical role of Asp206 stabilizing residues on the catalytic mechanism of the Ideonella sakaiensis PETase

“…This general approach has been used with success in the detailed atomic-level study of the catalytic mechanism of several enzymes. 64–69…”

“…This general approach has been used with success in the detailed atomic-level study of the catalytic mechanism of several enzymes. [64][65][66][67][68][69] Activation and reaction Gibbs free energies for each reaction step were determined by the difference between the Gibbs free energies of TS and reactant, or product and reactant, respectively.…”

A Proterozoic microbial origin of extant cyanide-hydrolyzing enzyme diversity

“…The physiological role of nitrilases has not been entirely elucidated, but the superfamily encompasses diverse functionality ranging from lipoprotein modification to nucleic acid catabolism and protein deamination ( Pace and Brenner, 2001 ; Howden and Preston, 2009 ; Thuku et al, 2009 ; Chhiba-Govindjee et al, 2019 ). Mammalian nitrilase homologs are associated with cell growth, vitamin synthesis, and tumor suppression ( Pace and Brenner, 2001 ; Barglow et al, 2008 ; Zhang et al, 2009 ; Silva Teixeira et al, 2021 ). Plant nitrilases play a role in hormone biosynthesis, nitrogen recycling, and detoxification of nitrile-containing compounds and intermediates ( Bestwick et al, 1993 ; Kobayashi et al, 1993 ; Kato et al, 2000 ; Piotrowski, 2008 ; Janowitz et al, 2009 ).…”