[2-H-3]ATP SYNTHESIS AND H-3 NMR-SPECTROSCOPY OF ENZYME-NUCLEOTIDE COMPLEXES - ADP AND ADP.VI BOUND TO MYOSIN SUBFRAGMENT-1

The synthesis of [2-H-3]ATP with specific activity high enough to use for H-3 NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substi tution at the 2-position and negligible tritium levels at other positi ons had a single H-3 NMR peak at 8.20 ppm in ID spectra obtained at 53 3 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes. The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity . In the presence of Mg2+, S1 converts [2-H-3]ATP to [2-H-3]ADP and th e complex S1.Mg[2-H-3]ADP has ADP bound in the active site. At 0-degre es-C, 1D H-3 NMR spectra of S1.Mg[2-H-3]ADP have two broadened peaks s hifted 0.55 and 0.90 ppm upfield from the peak due to free [2-H-3]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-H-3]ADP were ob tained in 180 min. The magnitude of the chemical shift caused by bindi ng is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkal i light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed prev iously by other techniques. Raising the temperature to 20-degrees-C ca uses small changes in the chemical shifts, narrows the peak widths fro m 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (V(i)) to produce S1.Mg[2-H-3]ADP.V(i ) shifts both peaks slightly more upfield without changing their width s or relative areas.