Kinetics of ATP and TNP-ATP binding to the active site of CheA from Thermotoga maritima.
The mechanism of nucleotide binding to the active site of Thermotoga maritima CheA was investigated using stopped-flow fluorescence experiments that monitored binding of ATP and TNP-ATP to the catalytic domain (P4) of CheA that had been engineered to include a tryptophan residue as a fluorescent reporter group at the active site (P4(F487W)). Rapid decreases in protein intrinsic fluorescence and increases in TNP-ATP fluorescence were observed during binding reactions, and time courses were analyzed to define the kinetic mechanisms for ATP and TNP-ATP binding. This analysis indicated that binding of ATP(Mg(2+)) to P4(F487W) involves a single reversible step with a k(on) of 0.92 +/- 0.09 microM(-1) s(-1), a k(off) of 1.9 +/- 0.4 s(-1), and a K(d) of 1.5-2.1 microM (all values determined at 4 degrees C). Binding of TNP-ATP(Mg(2+)) to P4(F487W) involves a more complicated mechanism, requiring at least three sequential steps. Computer simulations and nonlinear regression analysis were used to estimate the rate constants of the forward and reverse reactions for each of the three steps in the reaction scheme [Formula: see text] Similar analysis indicated that an alternative reaction scheme, involving a rate-limiting conformational change in P4 prior to TNP-ATP binding, did an equally good job of accounting for all of the kinetics results:[Formula: see text] In both models, steps 2 and 3 have slow reversal rates that contribute to the high affinity of the active site for TNP-ATP (K(d) = 0.015 microM). These results highlight the dramatic effect of the TNP moieties on CheA-nucleotide interactions, and they provide the first detailed information about the kinetic mechanism underlying interaction of a protein histidine kinase with this tight-binding inhibitor.
Eaton
Anna K
AK
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, USA.
Stewart
Richard C
RC
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D001665:Binding Sites / Q000235:genetics; D000071677:Histidine Kinase; D007700:Kinetics; D011494:Protein Kinases; D020124:Thermotoga maritima / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000235:genetics* / Q000378:metabolism
C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D000071677:Histidine Kinase