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 approximately 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 approximately 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 (Kd approximately 10(3)-fold higher than that of GDP) and dramatic discrimination against ADP or ATP (Kd approximately 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.
Simon
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Abteilung Physikalische Biochemie, Max-Planck-Institut für Molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund.
Zerial
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Goody
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D002474:Cell-Free System; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins
D017931:DNA Primers; D011994:Recombinant Proteins; D000090802:rab7 GTP-Binding Proteins; D006153:Guanosine Diphosphate; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D008274:Magnesium