Monitoring the real-time kinetics of the hydrolysis reaction of guanine nucleotide-binding proteins.
The conversion of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (Pi) by guanine nucleotide-binding proteins (GNBPs) is a fundamental enzyme reaction in living cells that acts as an important timer in a variety of biological processes. This reaction is intrinsically slow but can be stimulated by GTPase-activating proteins (GAPs) by several orders of magnitude. In the present study, we synthesized and characterized a new fluorescent nucleotide, 2'(3')-O-(N-ethylcarbamoyl-(5''-carboxytetramethylrhodamine) amide)-GTP, or tamraGTP, which is sensitive towards conformational changes of certain GNBPs induced by GTP hydrolysis. Unlike other fluorescent nucleotides, tamra-GTP allows real-time monitoring of the kinetics of the intrinsic and GAP-catalyzed GTP hydrolysis reactions of small GNBPs from the Rho family.
Eberth
Alexander
A
Department of Structural Biology, Max-Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany.
Dvorsky
Radovan
R
Becker
Christian F W
CF
Beste
Andrea
A
Goody
Roger S
RS
Ahmadian
Mohammad Reza
MR
D001665:Binding Sites; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D020830:rac1 GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D005456:Fluorescent Dyes; C508041:tamraGTP; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins