The mechanism of GTP hydrolysis by dynamin II: a transient kinetic study.
Dynamin II is a 98 kDa protein (870 amino acids) required for the late stages of clathrin-mediated endocytosis. The GTPase activity of dynamin is required for its function in the budding stages of receptor-mediated endocytosis and synaptic vesicle recycling. This activity is stimulated when dynamin self-associates on multivalent binding surfaces, such as microtubules and anionic liposomes. We first investigated the oligomeric state of dynamin II by analytical ultracentrifuge sedimentation equilibrium measurements at high ionic strength and found that it was best described by a monomer-tetramer equilibrium. We then studied the intrinsic dynamin GTPase mechanism by using a combination of fluorescence stopped-flow and HPLC methods using the fluorescent analogue of GTP, mantdGTP (2'-deoxy-3'-O-(N-methylanthraniloyl) guanosine-5'-triphosphate), under the same ionic strength conditions. The results are interpreted as showing that mantdGTP binds to dynamin in a two-step mechanism. The dissociation constant of mantdGTP binding to dynamin, calculated from the ratio of the off-rate to the on-rate (k(off)/k(on)), was 0.5 microM. Cleavage of mantdGTP then occurs to mantdGDP and P(i) followed by the rapid release of mantdGDP and P(i). No evidence of reversibility of hydrolysis was observed. The cleavage step itself is the rate-limiting step in the mechanism. This mechanism more closely resembles that of the Ras family of proteins involved in cell signaling than the myosin ATPase involved in cellular motility.
Binns
D D
DD
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041, USA.
Helms
M K
MK
Barylko
B
B
Davis
C T
CT
Jameson
D M
DM
Albanesi
J P
JP
Eccleston
J F
JF
D000818:Animals; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D034281:Dynamins; D005456:Fluorescent Dyes; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D014461:Ultracentrifugation; D062367:ortho-Aminobenzoates
D005456:Fluorescent Dyes; D062367:ortho-Aminobenzoates; C065197:3'-(methylanthraniloyl)-2'-deoxy-guanosine diphosphate; D006153:Guanosine Diphosphate; C063981:2'(3')-O-(N-methyl)anthraniloylguanosine 5'-triphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D034281:Dynamins