A theoretical study on the catalytic mechanism of Mus musculus adenosine deaminase

Abstract

The catalytic mechanism of Mus musculus adenosine deaminase (ADA) has been studied by quantum mechanics and two-layered ONIOM calculations. Our calculations show that the previously proposed mechanism, involving His238 as the general base to activate the Zn-bound water, has a high activation barrier of about 28 kcal/mol at the proposed rate-determining nucleophilic addition step, and the corresponding calculated kinetic isotope effects are significantly different from the recent experimental observations. We propose a revised mechanism based on calculations, in which Glu217 serves as the general base to abstract the proton of the Zn-bound water, and the protonated Glu217 then activates the substrate for the subsequent nucleophilic addition. The rate-determining step is the proton transfer from Zn-OH to 6-NH(2) of the tetrahedral intermediate, in which His238 serves as a proton shuttle for the proton transfer. The calculated kinetic isotope effects agree well with the experimental data, and calculated activation energy is also consistent with the experimental reaction rate.

Moonlighting Adenosine Deaminase: A Target Protein for Drug Development

“…The proposed catalytic mechanism for ADA reaction was mainly based on the combination of results from crystallographic studies, site‐directed mutagenesis experiments, and quantum and molecular mechanics studies . Adenosine deamination can be viewed as follows: first, by acting as a powerful electrophile, Zn 2+ activates the catalytic water molecule creating a hydroxide nucleophile.…”

“…For others, the general base is Asp 295 and the role of His 238 is to assist in orienting the water molecule and stabilizing the charge of the attacking negative hydroxylate via its positive charge . Wu et al proposed a revised mechanism in which Glu 217 serves as the general base to abstract the proton from the Zn 2+ ‐bound water. Then, the protonated Glu 217, located coplanar with the purine ring, activates the substrate for the subsequent nucleophilic addition (see Fig.…”

“…The stereospecificity of the chemical reaction is conferred by the location of the Zn 2+ , His 238, and Asp 295 on the B‐face side of the purine ring . Using quantum mechanics and two‐layered ONIOM calculations, Wu et al showed that the proton transfer from Zn 2+ ‐OH to 6‐NH 2 in the tetrahedral intermediate is the rate‐limiting process, in which His 238 serves as an important proton shuttle for the proton transfer. In the final step, which is not completely understood, the C6 amino group abstracts a proton to form the ammonia leaving group and the inosine product, and so the tetrahedral intermediate collapses.…”

“…Schematic diagram of transition‐state intermediates of ADA‐catalyzed reaction. The transition structure TS 1b of the mechanism B of Wu et al is shown in (a). The tetrahedral intermediate proposed by Sideraki et al is shown in (b).…”

“…The tetrahedral intermediate proposed by Sideraki et al is shown in (b). Extracted and adapted from the originals …”