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

Abstract

The catalytic mechanism of Pdx2 was studied with atomic detail employing the computational ONIOM hybrid QM/MM methodology. Pdx2 employs a Cys‐His‐Glu catalytic triad to deaminate glutamine to glutamate and ammonia – the source of the nitrogen of pyridoxal 5’‐phosphate (PLP). This enzyme is, therefore, a rate‐limiting step in the PLP biosynthetic pathway of Malaria and Tuberculosis pathogens that rely on this mechanism to obtain PLP. For this reason, Pdx2 is considered a novel and promising drug target to treat these diseases. The results obtained show that the catalytic mechanism of Pdx2 occurs in six steps that can be divided into four stages: (i) activation of Cys87, (ii) deamination of glutamine with the formation of the glutamyl‐thioester intermediate, (iii) hydrolysis of the formed intermediate, and (iv) enzymatic turnover. The kinetic data available in the literature (19.1–19.5 kcal mol−1) agree very well with the calculated free energy barrier of the hydrolytic step (18.2 kcal.mol−11), which is the rate‐limiting step of the catalytic process when substrate is readily available in the active site. This catalytic mechanism differs from other known amidases in three main points: i) it requires the activation of the nucleophile Cys87 to a thiolate; ii) the hydrolysis occurs in a single step and therefore does not require the formation of a second tetrahedral reaction intermediate, as it is proposed, and iii) Glu198 does not have a direct role in the catalytic process. Together, these results can be used for the synthesis of new transition state analogue inhibitors capable of inhibiting Pdx2 and impair diseases like Malaria and Tuberculosis.

Catalytic Effects of Active Site Conformational Change in the Allosteric Activation of Imidazole Glycerol Phosphate Synthase

“…The hydrolysis reaction mechanism has not yet been studied at the atomic level in IGPS, to the best of our knowledge. However, similar well-studied enzymes, such as other class-I GATs and cysteine/serine proteases, provide a useful theoretical foundation. ,,− These reactions are proposed to occur via two stages: acylation and deacylation. In the acylation stage, the nucleophilic h Cys84 attacks glutamine to form a glutamyl thioester, first structurally observed in another class-I GAT, carbamoyl phosphate synthetase, via a tetrahedral oxyanion intermediate.…”

A Molecular Mechanics Energy Partitioning Software for Biomolecular Systems

“…Molecular mechanics (MM) [ 1 ] has been widely used in computational studies [ 2 , 3 , 4 , 5 ], particularly in large molecular systems in which more accurate methods, e.g., quantum mechanics (QM), cannot be employed. For example, in the study of enzymatic mechanisms [ 3 , 6 , 7 , 8 ], MM Hamiltonians are employed in MM minimizations, MD simulations, and QM/MM calculations [ 9 ].…”