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Enzymatic analysis of EF‐Tu and EF‐Ts from Pseudomonas aeruginosa
Author(s) -
Tran Alexis,
Palmer Stephanie,
Bullard James
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.986.4
Subject(s) - gtp' , chemistry , dissociation constant , ef tu , ternary complex , enzyme , stereochemistry , transfer rna , ribosome , biochemistry , crystallography , rna , receptor , gene
Elongation factors (EF) facilitate many steps in the process of the biosynthesis of proteins. EF‐Tu in particular plays a central role in the GTP‐dependent placement of aminoacylated tRNA at the A site of the ribosome. EF‐Ts acts by catalyzing the change of EF‐Tu from a GDP‐bound inactive state to a GTP‐bound active state. We present here analysis of the kinetic parameters of EF‐Tu and EF‐Ts. Results Kinetic parameters for the interaction of EF‐Tu with its substrate GDP in the absence of EF‐Ts were observed to be: K M = 33 mM, k cat = 0.003 sec −1 and the specificity constant k cat / K M was then calculated as 0.1 × 10 −3 sec −1 uM −1 . In the presence of EF‐Ts, these values were shifted to: K M = 2 mM, k cat = 0.005 sec −1 and the specificity constant k cat / K M was then calculated as 2.5 × 10 −3 sec −1 uM −1 . We determined the equilibrium dissociation constant ( K GDP ) to be in the range of 100–160 nM in the absence of EF‐Ts at various temperatures. The presence of EFTs stimulated the exchange of GDP by EF‐Tu by up to 10‐fold. P. aeruginosa EF‐Tu was also shown to be active in forming a ternary complex with GTP and aminoacylated tRNA and was functional in polyU dependent placement of Phe‐tRNA Phe at the A site of the P. aeruginosa ribosome. Conclusion EF‐Tu and EF‐Ts identified in P. aeruginosa were cloned, expressed and purified and shown to be functional in assays suggesting that each is functional in protein synthesis. Research was supported by NIH grant 1SC3GM098173–01A1.