Active-Site Assembly in Glutaminyl-tRNA Synthetase by tRNA-Mediated Induced Fit

Abstract

Structure-based mutational analysis was employed to probe an unusual intramolecular interaction between partially buried glutamate residues adjacent to the active site of Escherichia coli glutaminyltRNA synthetase (GlnRS). Crystal structures of unliganded GlnRS and of the GlnRS-tRNA Gln complex reveal that the Glu34 and Glu73 side-chain carboxylates contact each other only in the tRNA-bound state, and that the interaction is formed via mutual induced-fit transitions that occur en route to the ground-state Michaelis complex. Steady-state and transient kinetic analysis of mutant enzymes suggest that formation of this intermolecular contact is a key event that facilitates proper formation of the active site. Mutants at both positions destabilize the binding of substrate glutamine at the opposite side of the active-site cleft, while Glu73 appears to play an additional important role by promoting correct binding of the 3′-acceptor end of tRNA adjacent to both ATP and glutamine. The data suggest the existence of multiple structural pathways by which the binding of tRNA propagates conformational transitions leading to proper formation of the glutamine binding site. The single-turnover kinetic analysis also establishes that the Glu34 carboxylate does not play a direct enzymatic role as a catalytic base to help deprotonate the tRNA-A76 nucleophilic 2′-hydroxyl group. The elimination of this previously proposed mechanism, together with recent chemical modification experiments in the histidyl-tRNA synthetase system, emphasizes that substrate-assisted catalysis by the phosphate of the aminoacyl adenylate may be a common means by which all tRNA synthetases facilitate the aminnoacyl transfer step of the reaction. KeywordsRNA-protein interaction; induced fit; aminoacyl-tRNA synthetase; enzyme catalysis Aminoacyl tRNA-synthetases comprise an ancient class of enzymes that are crucial to maintaining fidelity in the genetic code. They catalyze specific aminoacyl-tRNA synthesis in a two-step reaction. First, energy from ATP hydrolysis is utilized to generate an activated aminoacyl adenylate intermediate possessing a mixed anhydride linkage (1). In a second step, the 2′ or 3′ ribose oxygen of the 3′-terminal A76 tRNA nucleotide attacks the carbonyl carbon atom of this linkage, forming aminoacyl-tRNA with release of AMP. The tRNA synthetases are divided into two classes possessing topologically distinct active-site domains (2). Other domains in the structure are primarily involved in discriminating against noncognate tRNAs, and diverge significantly among the enzymes.

Structural dynamics of the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase

“…The reaction is most likely substrate assisted, with the phosphate of the adenylate acting as the general base to abstract the proton from the 2′OH in a concerted mechanism 14,36 (Fig. 4c).…”

Amino acid discrimination by arginyl‐tRNA synthetases as revealed by an examination of natural specificity variants

“…For glutamyl-tRNA synthetase, the presence of tRNA eliminates non-specific binding of d-glutamic acid and l-aspartic acid to the enzyme [9,10]. Detailed analysis of glutaminyl-tRNA synthetase has led to the proposal of an induced-fit type of active site rearrangement that plays a role in enzyme specificity [11][12][13], and the concept of discriminatory elements in tRNA that participate in amino acid selection has been proposed [14]. It would then be consistent with our observations for jack bean tRNA Arg to trigger an active site rearrangement in the jack bean enzyme that provides the means to enhance amino acid discrimination.…”

“…For others that are specific for structurally idiosyncratic amino acids, no active editing may be required. In the case of glutamyl‐ and glutaminyl‐tRNA synthetases, which together with arginyl‐tRNA synthetase form a subgroup of enzymes that require tRNA for amino acid activation, the potential for misrecognition of related amino acids has been investigated [9–13] and modulated by amino acid replacements and active site redesign [14]. A mechanism that does not rely on hydrolytic editing but resembles an induced fit type of substrate selection, including the participation of tRNA structural features, has been proposed [14].…”

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

“…16 Based on their observed dissimilar active sites and apparent common utilization of a non-bridging phospho-oxygen of the substrate as the mechanistic base, it has been suggested that for aaRS's such a substrate-assisted catalysis (SAC) mechanism may be a general feature of these presumably ancient enzymes. 5,16,22–26 …”