Elucidation of the Reaction Mechanism of Cavia porcellus l-Asparaginase: A QM/MM Study

Abstract

The L-asparaginase (L-ASNase) enzyme catalyzes the conversion of the non-essential amino acid L-asparagine into Laspartic acid and ammonia. Importantly, the L-ASNases are used as a key part of the treatment of acute lymphoblastic leukemia (ALL); however, despite their benefits, they trigger severe side effects because they have their origin in bacterial species (Escherichia coli and Erwinia chrysanthemi). Therefore, one way to solve these side effects is the use of L-ASNases with characteristics similar to those of bacterial types, but from different sources. In this sense, Cavia porcellus L-ASNase (CpA) of mammalian origin is a promising enzyme because it possesses similarities with bacterial species. In this work, the hydrolysis reaction for C. porcellus L-asparaginase was studied from an atomistic point of view. The QM/MM methodology was employed to describe the reaction, from which it was found that the conversion mechanism of L-asparagine into L-aspartic acid occurs in four steps. It was identified that the nucleophilic attack and release of the ammonia group is the ratelimiting step of the reaction. In this step, the nucleophile (Thr 19 ) attacks the substrate (ASN) leading to the formation of a covalent intermediate and release of the leaving group (ammonia). The calculated energy barrier is 18.9 kcal mol −1 , at the M06-2X+D3(0)/6-311+G(2d,2p)//CHARMM36 level of theory, which is in agreement with the kinetic data available in the literature, 15.9 kcal mol −1 (derived from the k cat value of 38.6 s −1 ). These catalytic aspects will hopefully pave the way toward enhanced forms of CpA. Finally, our work emphasizes that computational calculations may enhance the rational design of mutations to improve the catalytic properties of the CpA enzyme.

Elucidation of the Active Form and Reaction Mechanism in Human Asparaginase Type III Using Multiscale Simulations

“…To describe the QM subsystem, we used the B3LYP functional , with D3 dispersion corrections and the 6-31+G* basis set. The B3LYP functional has been selected based on the previous works done on ASNases I and II, − where this functional was found to provide energies in good agreement with the experimental data. Preliminary calculations used to explore different mechanistic proposals were made at the DFTB3/MM level .…”

“…However, no significant progress was observed in regard to the diminution of the immunogenetic response . In order to overcome these side effects due to the bacterial origin of the enzymes used in ALL treatments, several trials have been oriented toward their substitution by l -ASNases of mammalian origins, such as human l -ASNases (hASNaseIII) , and guinea pig ASNase (gpASNaseIII). , While several theoretical studies have been performed on the reaction mechanism of l -ASNase type I and II, − to our knowledge, no research has yet been done on the mechanism of ASNasesIII.…”

Elucidation of the catalytic mechanism of a single-metal dependent homing endonuclease using QM and QM/MM approaches: the case study of I-PpoI

“…1a) as calculations initiated from an X-ray crystal structure have been successfully used to elucidate the reaction mechanisms for several enzymes. [82][83][84][85][86] The alanine mutation at position 98 in the crystal structure was manually reverted to H98 using PyMOL based on the residue orientation in the crystal structure of the wild type I-PpoI PC (1.80 Å resolution; PDB ID: 1A73, Fig. 1b).…”