Interaction of N-tosyl-L-phenylalanylchloromethane with Thermus thermophilus elongation factor Tu.
The interaction of N-tosyl-L-phenylalanylchloromethane (TosPheCH2Cl) with Thermus thermophilus elongation factor Tu (EF-Tu) was studied by affinity labelling and NMR spectroscopy. TosPheCH2Cl binds to GDP and GTP conformers of EF-Tu. The interaction of TosPheCH2Cl with EF-Tu x GDP leads to alkylation of Cys82, while interaction of TosPheCH2Cl with EF-Tu x GTP does not lead to covalent labelling. [A82]EF-Tu, in which the Cys82 is replaced by Ala, has similar properties to wild-type EF-Tu with respect to GTPase activity, binding of guanine nucleotides, interaction with elongation factor Ts (EF-Ts) and interaction with ribosomes. This structural change did not lead to changes, compared with wild-type EF-Tu in the functionality of [A82]EF-Tu, either in the GTP or in the GDP conformation. TosPheCH2Cl binds to EF-Tu x GTP with a dissociation constant of 10 microM. The interaction of TosPheCH2Cl with EF-Tu promotes the hydration of the carbonyl group of TosPheCH2Cl. TosPheCH2Cl competes with aminoacyl-tRNA for its binding site on EF-Tu x GTP. Covalent modification of Cys82 by TosPheCH2Cl does not prevent nucleotide binding and GTPase activity, but interferes with the interaction with aminoacyl-tRNA. TosPheCH2Cl probably mimics the aminoacyl residue of the aminoacyl-tRNA and binds to its binding site on EF-Tu x GTP. This rather specific interaction with EF-Tu x GTP does not allow the modification of Cys82, whereas the loose interaction of TosPheCH2Cl with EF-Tu x GDP leads to alkylation of this residue.
Nawrot
B
B
Laboratorium für Biochemie, Universität Bayreuth, Germany.
Hillenbrand
R
R
Limmer
S
S
Grillenbeck
N
N
Sprinzl
M
M
D000345:Affinity Labels; D001665:Binding Sites; D006160:Guanosine Triphosphate / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D010444:Peptide Elongation Factor Tu / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016963:Thermus thermophilus / Q000737:chemistry*; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000378:metabolism / Q000494:pharmacology*
D000345:Affinity Labels; D014108:Tosylphenylalanyl Chloromethyl Ketone; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu