The α-Amino Group of the Threonine Substrate as The General Base During tRNA Aminoacylation: A New Version of Substrate-Assisted Catalysis Predicted by Hybrid DFT

Abstract

Density functional theory-based methods in combination with large chemical models have been used to investigate the mechanism of the second half-reaction catalyzed by Thr-tRNA synthetase; aminoacyl transfer from Thr-AMP onto the A763'OH of the cognate tRNA. In particular, we have examined pathways in which an active site His309 residue is either protonated or neutral (i.e., potentially able to act as a base). In the protonated His309-assisted mechanism, the rate-limiting step is formation of the tetrahedral intermediate. The barrier for this step is 155.0 kJ mol−1 and thus, such a pathway is concluded to not be enzymatically feasible. For the neutral His309-assisted mechanism two models were used with the difference being whether Lys465 was included. For either model the barrier of the rate-limiting step is below the upper-thermodynamic enzymatic limit of ∼125 kJ mol−1. Specifically, without Lys465 the rate-limiting barrier is 122.1 kJ mol−1 and corresponds to a rotation about the tetrahedral intermediates Ccarb—OH bond. For the model with Lys465 the rate-limiting barrier is slightly lower and corresponds to the formation of the tetrahedral intermediate. Importantly, for both neutral His309’ models the neutral amino group of the threonyl substrate directly acts as the proton accepter; in the formation of the tetrahedral intermediate the A763'OH proton is directly transferred onto the Thr-NH2. Therefore, the overall mechanism follows a general substrate assisted catalytic mechanism.

Roles of the Active Site Zn(II) and Residues in Substrate Discrimination by Threonyl-tRNA Synthetase: An MD and QM/MM Investigation

“…As part of an earlier QM-cluster-based computational study on ThrRS, we examined the Michaelis complex and aminoacylation mechanism of ThrRS. 21 It was concluded that, in the pre-reactive Michaelis complex, the threonyl moiety of the Thr-AMP substrate was bidentately ligated to the Zn(II) ion via both its neutral α-NH 2 and side chain β-OH groups, giving a pentacoordinate Zn(II) center. In the corresponding reactive complex (RC), however, the substrate's threonyl moiety was only monodentately ligated to the Zn(II) via its side chain βhydroxy oxygen.…”

Multi-Scale Computational Enzymology: Enhancing Our Understanding of Enzymatic Catalysis

“…They are perhaps best known for their central role in protein synthesis. Specifically, they catalyze the aminoacylation of their cognate tRNA [ 56 – 59 ] via the two half reactions shown in Scheme 4 [ 59 – 61 ]. First, the amino acid is activated by reacting with ATP to give an aminoacyladenylate (aaAMP).…”

“…Recently, we have used several computational methods such as MD and QM-cluster approach in a complementary fashion to elucidate the nature of the active site base in the second half reaction of theronyl-tRNA [ 59 , 62 ]. Unfortunately, while several X-ray structures are available for ThrRS, none have both ThrAMP and tRNA Thr bound [ 62 ].…”

Substrate-Assisted and Enzymatic Pretransfer Editing of Nonstandard Amino Acids by Methionyl-tRNA Synthetase

“…7,8 These reactions are thought to proceed via substrate-assisted mechanisms in which the substrates themselves catalyze their transfer onto the tRNA aa . 9,10 Central to their physiological roles, however, is the difficult yet essential task of discriminating between amino acids. Indeed, it has been suggested that the error rate in translation cannot exceed 10 −4 for proper growth and function.…”

“…For example, ThrRS must discern its substrate threonine from serine and valine. 9,13,14 This matter is further complicated as aaRSs must also discriminate against other species such as the nonstandard amino acids homocysteine (Hcy) and homoserine (Hse). 15−17 The latter are highly reactive, and their incorporation into proteins has been implicated in a number of diseases, including stroke, cancer, and Alzheimer's.…”