Aminoacetylation Reaction Catalyzed by Leucyl-tRNA Synthetase Operates via a Self-Assisted Mechanism Using a Conserved Residue and the Aminoacyl Substrate.
Leucyl-tRNA synthetase catalyzes attachment of leucine amino acid to its cognate tRNA. During the second, aminoacetylation, step of the reaction, the leucyl moiety is transferred from leucyl-adenylate to the terminal A76 adenosine of tRNA. In this work, we have investigated the aminoacetylation step catalyzed by leucyl-tRNA synthase, using ab initio quantum chemical/molecular mechanical hybrid potentials in conjunction with reaction-path-location algorithms and molecular dynamics free energy simulations. We have modeled reaction mechanisms arising from both crystallographic studies and computational work. We invoke various groups as potential proton acceptors-namely, the phosphate and leucyl amino groups of leucyl-adenylate, the A76 base of tRNA, and the Asp80 and Glu532 residues of the protein-and consider both metal-assisted and metal-free reactions. Free energy calculations indicate that both the phosphate group of leucyl adenylate and Glu532 are not strong bases. This agrees with the results of the quantum chemical/molecular mechanical reaction path calculations which give high free energy barriers for the studied pathways involving these groups. A self-assisted mechanism with the leucyl amino group and Asp80 as proton acceptors is the most likely. Furthermore, in this mechanism the presence of a metal ion coordinated by the phosphate group and Glu532 strongly activates the reaction.
Laboratoire de Biochimie, UMR 7654, Ecole Polytechnique, CNRS , F-91128 Palaiseau Cedex, France.
Alexey
A
Aleksandrov
European Molecular Biology Laboratory (EMBL), Grenoble Outstation and Unit of Virus Host-Cell Interactions, University of Grenoble-EMBL-CNRS , 38044 Grenoble, France.
Andrés
A
Palencia
European Molecular Biology Laboratory (EMBL), Grenoble Outstation and Unit of Virus Host-Cell Interactions, University of Grenoble-EMBL-CNRS , 38044 Grenoble, France.
Stephen
S
Cusack
Dynamo Team, DYNAMOP Group, UMR 5075, Université Grenoble 1, CNRS, CEA, Institut de Biologie Structurale , 71 Avenue des Martyrs, CS 10090, 38044 Grenoble Cedex 9, France.
Martin
M
Field
D000107:Acetylation; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D007935:Leucine-tRNA Ligase / Q000737:chemistry / Q000378:metabolism*; D008670:Metals / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D017434:Protein Structure, Tertiary; D011789:Quantum Theory; D013379:Substrate Specificity; D013816:Thermodynamics; D014867:Water / Q000737:chemistry / Q000378:metabolism
D008670:Metals; D014867:Water; D007935:Leucine-tRNA Ligase