Conformational change rate-limits GTP hydrolysis: the mechanism of the ATP sulfurylase-GTPase.
The fluorescent GTP analogues 3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-(beta, gamma-imidotriphosphate) (mGMPPNP) and 3'-O-(N-methylanthraniloyl)-2'-deoxy-GTP (mGTP) were used to demonstrate that an enzyme isomerization precedes and rate-limits beta,gamma-bond cleavage in the catalytic cycle of the ATP sulfurylase-GTPase, from E. coli K-12. The binding of mGMPPNP to the E.AMP.PPi complex of ATP sulfurylase is biphasic, indicating that an isomerization occurs in the binding reaction. The isomerization mechanism was assigned based on the results of the enzyme concentration dependence of the observed rate constants, kobs, for both phases of the binding reaction, and sequential-mixing, nucleotide release experiments. The isomerization occurs after, and is driven by, the addition of mGMPPNP. Values were determined for each of the rate constants associated with the two-step kinetic model used in the interpretation of the results. A comparison of the enzyme concentration dependence of kobs for the hydrolysis and binding reactions reveals that the rate constants for the corresponding steps of these two reactions are extremely similar. The virtually identical rate constants for isomerization and beta, gamma-bond scission strongly suggest that isomerization rate-limits bond breaking. The implications of these finding for GTPase/target interactions and the mechanism of energetic linkage in the ATP sulfurylase system are discussed.
Wei
J
J
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Leyh
T S
TS
D000250:Adenosine Phosphosulfate / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000737:chemistry / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D013430:Sulfate Adenylyltransferase / Q000737:chemistry / Q000378:metabolism*
D020033:Protein Isoforms; D000250:Adenosine Phosphosulfate; D006160:Guanosine Triphosphate; D013430:Sulfate Adenylyltransferase; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu