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 fluoresence and fluorescence reson ance energy transfer show that nucleotide binding is in general at lea st a two-step process. The first step is a weak initial binding, which is followed by a relatively slow isomerization of the protein-nucleot ide complex in which changes of both intrinsic and extrinsic fluoresce nce, as well as energy transfer, occur. The values obtained for the eq uilibrium and kinetic constants confirm the earlier observation that E F-Tu has a higher affinity for GDP than GTP. This is mainly due to a l ower dissociation rate constant for GDP, in combination with a somewha t higher effective association rate constant. Modifications of the tri phosphate moiety of GTP are quite well tolerated by EF-Tu, with GTP ga mma S displaying the same affinity as GTP and with GppNHp and GppCH(2) p being only ca. 2-3-fold less strongly bound. Caged GTP is bound abou t 6-fold more weakly than GTP. These results suggest that the binding of GppNHp and GppCH(2)p is likely to be similar to that of GTP. The ph otolytic protecting group of caged GTP (or the loss of one of the nega tive charges on the gamma-phosphate group) appears to interfere to a c ertain extent with the interaction with the protein, but the affinity is high enough to permit generation of 1:1 complexes for dynamic struc tural studies. Discrimination between GDP and ADP is dramatic, with a difference of 6 orders of magnitude in affinity.