Interaction of guanosine nucleotides and their analogs with elongation factor Tu from Thermus thermophilus.
Transient kinetic experiments on the interaction of nucleotide-free EF-Tu from Thermus thermophilus with nucleotides using intrinsic protein fluorescence, extrinsic nucleotide fluorescence and fluorescence resonance energy transfer show that nucleotide binding is in general at least a two-step process. The first step is a weak initial binding, which is followed by a relatively slow isomerization of the protein-nucleotide complex in which changes of both intrinsic and extrinsic fluorescence, as well as energy transfer, occur. The values obtained for the equilibrium and kinetic constants confirm the earlier observation that EF-Tu has a higher affinity for GDP than GTP. This is mainly due to a lower dissociation rate constant for GDP, in combination with a somewhat higher effective association rate constant. Modifications of the triphosphate moiety of GTP are quite well tolerated by EF-Tu, with GTP gamma S displaying the same affinity as GTP and with GppNHp and GppCH2p being only ca. 2-3-fold less strongly bound. Caged GTP is bound about 6-fold more weakly than GTP. These results suggest that the binding of GppNHp and GppCH2p is likely to be similar to that of GTP. The photolytic protecting group of caged GTP (or the loss of one of the negative charges on the gamma-phosphate group) appears to interfere to a certain extent with the interaction with the protein, but the affinity is high enough to permit generation of 1:1 complexes for dynamic structural studies. Discrimination between GDP and ADP is dramatic, with a difference of 6 orders of magnitude in affinity.
Wagner
A
A
Laboratorium für Biochemie, Universität Bayreuth, Germany.
Simon
I
I
Sprinzl
M
M
Goody
R S
RS
D000244:Adenosine Diphosphate / Q000737:chemistry; D006150:Guanine Nucleotides / Q000737:chemistry*; D007700:Kinetics; D010444:Peptide Elongation Factor Tu / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D016963:Thermus thermophilus / Q000737:chemistry*
D006150:Guanine Nucleotides; D000244:Adenosine Diphosphate; D010444:Peptide Elongation Factor Tu