Kinetics of interaction of Rab5 and Rab7 with nucleotides and magnesium ions

We describe here the kinetics of the interaction of GTP and GDP with the small GTP-binding proteins Rab5 and Rab7. It was possible to make use of the intrinsic fluorescence of these proteins, since Rab5 contains two and Rab7 three tryptophan residues, respectively, With both enzymes, there is a significant decrease in fluorescence on binding GTP and an increase on binding GDP, As with the small GTP-binding protein Ha-Ras p21 and with EF-Tu, nucleotide binding occurs in at least two steps and is describable in terms of a relatively weak initial interaction followed by a highly irreversible isomerization of the protein-nucleotide complex, which results in a change in the fluorescence properties, Dissociation of GDP and GTP could be followed in a time-dependent manner using fluorescently labeled GDP (methylanthraniloyl GDP) as displacing agent and taking advantage of substantial fluorescent energy transfer from tryptophan to the nucleotide. Fluorescence techniques could also be used to quantitate the interaction of Mg2+ ions with the GTP and GDP forms of Rab7, and it was shown that the metal ion was bound similar to 1000-fold more strongly to the GTP than the GDP form. The rate of GTP cleavage by the two proteins differed by a factor of similar to 20 (2 x 10(-3) s(-1) for Rab5 and 9 x 10(-4) s(-1) for Rab7 at 37 degrees C), Both proteins showed significant discrimination against xanthosine 5'-O-diphosphate (K-d similar to 10(3)-fold higher than that of GDP) and dramatic discrimination against ADP or ATP (K-d similar to 10(6)-fold higher than that of GDP), The results demonstrate a high degree of mechanistic similarity between the Rab proteins and other GTP-binding proteins, which have been examined in detail, including Ha-Ras p21, Ran, and EF-Tu.