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A Leucyl-tRNA Synthetase Urzyme: Authenticity of tRNA Synthetase Catalytic Activities and Promiscuous Phosphorylation of Leucyl-5′AMP
- Source :
- International Journal of Molecular Sciences; Volume 23; Issue 8; Pages: 4229
- Publication Year :
- 2022
- Publisher :
- MDPI AG, 2022.
-
Abstract
- Aminoacyl-tRNA synthetase (aaRS)/tRNA cognate pairs translate the genetic code by synthesizing specific aminoacyl-tRNAs that are assembled on messenger RNA by the ribosome. Deconstruction of the two distinct aaRS superfamilies (Classes) has provided conceptual and experimental models for their early evolution. Urzymes, containing ~120–130 amino acids excerpted from regions where genetic coding sequence complementarities have been identified, are key experimental models motivated by the proposal of a single bidirectional ancestral gene. Previous reports that Class I and Class II urzymes accelerate both amino acid activation and tRNA aminoacylation have not been extended to other synthetases. We describe a third urzyme (LeuAC) prepared from the Class IA Pyrococcus horikoshii leucyl-tRNA synthetase. We adduce multiple lines of evidence for the authenticity of its catalysis of both canonical reactions, amino acid activation and tRNALeu aminoacylation. Mutation of the three active-site lysine residues to alanine causes significant, but modest reduction in both amino acid activation and aminoacylation. LeuAC also catalyzes production of ADP, a non-canonical enzymatic function that has been overlooked since it first was described for several full-length aaRS in the 1970s. Structural data suggest that the LeuAC active site accommodates two ATP conformations that are prominent in water but rarely seen bound to proteins, accounting for successive, in situ phosphorylation of the bound leucyl-5′AMP phosphate, accounting for ADP production. This unusual ATP consumption regenerates the transition state for amino acid activation and suggests, in turn, that in the absence of the editing and anticodon-binding domains, LeuAC releases leu-5′AMP unusually slowly, relative to the two phosphorylation reactions.
- Subjects :
- Organic Chemistry
genetic coding
protein synthesis
mechanistic enzymology
protein engineering
evolutionary intermediates
validating weak catalytic activities
single turnover kinetics
structural biology
evolutionary changes in the occupation of sequence space
General Medicine
Adenosine Monophosphate
Catalysis
Computer Science Applications
Adenosine Diphosphate
Amino Acyl-tRNA Synthetases
Inorganic Chemistry
Adenosine Triphosphate
Leucine-tRNA Ligase
Amino Acids
Phosphorylation
Physical and Theoretical Chemistry
Molecular Biology
Spectroscopy
Subjects
Details
- ISSN :
- 14220067
- Volume :
- 23
- Database :
- OpenAIRE
- Journal :
- International Journal of Molecular Sciences
- Accession number :
- edsair.doi.dedup.....e373a1ff71cc1c31462bad79dd74fe95
- Full Text :
- https://doi.org/10.3390/ijms23084229