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  • RO-3 receptor In three groups independently proposed two dif

    2022-05-04

    In 2001, three groups independently proposed two different mechanisms for the catalytic reaction of GlxI [5], [6], [7]. Richter and Krauss (RK) used HF/4–31G calculations of the active site, coupled with a frozen effective fragment potential description [8], [9] of eleven residues in the binding site and proposed a three-step mechanism for the reaction of the S enantiomer of the substrate [5]. Himo and Siegbahn (HS) used density functional theory (DFT) and proposed a five-step mechanism for the S substrate [6] (the step counts exclude the binding of the substrate and the dissociation of the product). Creighton and Hamilton (CH) published a minireview, in which they discussed the catalytic reaction of GlxI as well as its stereospecificity [7]. They summarized experimental aspects of the catalytic mechanism of GlxI and suggested the same three-step mechanism as that proposed by RK. In addition, Åqvist and coworkers have also studied the first RO-3 receptor transfer with the empirical valence bond method [10]. Scheme 2 shows the mechanism for the S enantiomer of the substrate proposed by RK and CH. The reaction starts with abstraction of H1 by Glu-172 (see Scheme 2 for the numbering of the atoms). Then, H1 is transferred from Glu-172 to C2 and concurrently H2 is transferred from O1 to Glu-99. Finally, H2 is transferred from Glu-99 to O2 and the product is formed. The first step of this mechanism is also the first step of the mechanism proposed by HS. However, in the second step of the latter mechanism, H1 moves from Glu-172 to O2 (Scheme 3). Next, the resulting intermediate is transformed to an isoenergetic structure, in which H1 binds to O1 and H2 binds to O2, but the hydrogen bonds with Glu-99 and Glu-172 are kept (these hydrogen bonds are indicated by dotted lines in Scheme 3). After that, Glu-172 abstracts H1 from O1 and finally transfers it to C2. The most challenging part in the catalytic mechanism of GlxI is its stereospecificity and the reaction of the R substrate. The first step of the proposed mechanisms for the R substrate in the various works is same, viz. the abstraction of H1 by Glu-99 [4], [5], [6], [7]. However, the three groups proposed different steps for the subsequent reaction. RK suggested that in the second step, Glu-172 receives H2 and transfers it to C2, whereas H1 goes to O2 (Scheme 4). HS proposed that after the first step, H1 moves to O2. Then, Glu-172 abstracts H2 from O1 and transfers it to the si face of C2 to produce the product (Scheme 5). CH proposed a dissociative mechanism for the R substrate, i.e. that the enzyme first converts the R substrate to the S substrate (via a dissociation of a glutathionyl mercaptide ion) and then processes the S substrate (Scheme 6). Thus, despite all the previous studies, details of the reaction mechanism and the stereospecificity of GlxI are still unknown. In this paper, we investigate all proposed mechanisms for both the S and R substrates on an equal footing. We have used the quantum mechanical (QM) cluster approach, which has extensively been used to study catalytic mechanisms and structures of enzymes [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26]. In this approach, the most important residues are cut out from the active site of the enzyme. It reduces the number of considered atoms to 50–200, which makes it possible to study the reaction by DFT methods. In order to avoid that the model may change significantly from the crystal structure during the geometry optimization, some atoms are kept fixed at their crystal-structure positions. In addition, to account for the protein surrounding, continuum-solvation techniques are used, which model the surrounding as a homogenous polarizable medium, characterized by a dielectric constant. The choice of this constant is somewhat arbitrary, but ε=4 is usually considered to be a good representation of the protein surrounding [27], [28], [29], [30], [31], [32], [33].