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 t he small GTP-binding proteins Rab5 and Rab7. It was possible to make u se of the intrinsic fluorescence of these proteins, since Rab5 contain s two and Rab7 three tryptophan residues, respectively, With both enzy mes, there is a significant decrease in fluorescence on binding GTP an d 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 ste ps and is describable in terms of a relatively weak initial interactio n followed by a highly irreversible isomerization of the protein-nucle otide complex, which results in a change in the fluorescence propertie s, Dissociation of GDP and GTP could be followed in a time-dependent m anner using fluorescently labeled GDP (methylanthraniloyl GDP) as disp lacing agent and taking advantage of substantial fluorescent energy tr ansfer from tryptophan to the nucleotide. Fluorescence techniques coul d 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 r ate of GTP cleavage by the two proteins differed by a factor of simila r 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 x anthosine 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 t o 10(6)-fold higher than that of GDP), The results demonstrate a high degree of mechanistic similarity between the Rab proteins and other GT P-binding proteins, which have been examined in detail, including Ha-R as p21, Ran, and EF-Tu.