Moderate discrimination of REP-1 between Rab7 x GDP and Rab7 x GTP arises from a difference of an order of magnitude in dissociation rates.
The kinetics of the interaction of Rab7 with REP-1 have been investigated using the fluorescence of GDP and GTP analogs at the active site of Rab7. The results show that REP-1 has higher affinity for the GDP bound form of Rab7 (Kd=1 nM) than for the GTP bound form (Kd=20 nM). Both affinities should still be sufficient for the formation of stable complexes in the cell. The association reaction proceeds in two steps for the GDP bound form. The initial step is fast (k+1 = ca. 10[7] M[-1] s[-1]) and concentration dependent while the second represents a slow equilibration (k+2 + k-2 = 3.5 s[-1]) which has little effect on the overall equilibrium. The difference in affinity of the two nucleotide bound forms arises from a difference in dissociation rates (0.012 s[-1] for Rab7 x GDP and 0.2 s[-1] for Rab7 x GTP).
Alexandrov
K
K
Max-Planck Institute for Molecular Physiology, Department of Physical Biochemistry, Dortmund, Germany.
Simon
I
I
Iakovenko
A
A
Holz
B
B
Goody
R S
RS
Scheidig
A J
AJ
D048868:Adaptor Proteins, Signal Transducing; D019883:Alkyl and Aryl Transferases; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism*; D019204:GTP-Binding Proteins; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017368:Protein Prenylation / Q000502:physiology; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D020691:rab GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C513166:Chm protein, rat; D011994:Recombinant Proteins; D000090802:rab7 GTP-Binding Proteins; C000716466:rab7 GTP-binding proteins, rat; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019883:Alkyl and Aryl Transferases; D019204:GTP-Binding Proteins; D020691:rab GTP-Binding Proteins