PROBING THE NUCLEOTIDE-BINDING SITES OF AXONEMAL DYNEIN WITH THE FLUORESCENT NUCLEOTIDE ANALOG 2'(3')-O-(-N-METHYLANTHRANILOYL)-ADENOSINE 5'-TRIPHOSPHATE

MantATP [2'(3')-O-(-N-methylanthraniloyl)-adenosine 5'-triphosphate] w as employed as a fluorescence probe of the nucleotide-binding sites of dynein from sea urchin sperm flagella. MantATP binds specifically wit h enhanced fluorescence (similar to 2.2-fold), homogeneous lifetime (8 .4 ns), and high anisotropy (r similar to 0.38) to dynein and can be d isplaced by ATP and ADP added to the medium. The association constants of mantATP complexed with dynein were determined from anisotropy titr ation data. Using a multiple stepwise equilibrium model, the average v alues of the first two association constants are K-1 = 2.7 x 10(5) M-1 and K-2 = 1.8 x 10(4) M-1. This value of K-1 is 7-8 times higher than that found previously for unsubstituted ATP, whereas K2 is little cha nged [Mocz and Gibbons (1996) Biochemistry 35, 9204-9211]. The lower-a ffinity binding sites, K-3 and K-4, observed previously could not be s tudied with mantATP within the available protein concentrations. The a lpha and beta heavy chain subfractions have binding parameters similar to those of intact dynein. Formation of the stable ternary complex of mantATP with dynein and monomeric vanadate is accompanied by only a m oderate increase in the binding affinities. Oligomeric vanadate reduce s the binding affinities by similar to 50%. Addition of TritonX-100, m ethanol, or various salts changes the binding affinities by up to 50%, suggesting that the microenvironment of the nucleotide-binding sites involves significant contributions from both polar and apolar interact ions. The distinct affinities of the individual binding sites are cons istent with a physiological role in regulating nucleotide binding.