A NOVEL ADENOSINE-TRIPHOSPHATE ANALOG WITH A HEAVY-ATOM TO TARGET THENUCLEOTIDE-BINDING SITE OF PROTEINS

We have synthesized 2'-deoxy-2'-iodoadenosine-5'-triphosphate (2'-IATP ), a heavy-atom analog of adenosine-5'-triphosphate. This compound was made for X-ray structural studies to target the nucleotide site of AT P binding proteins. It was diffused successfully into crystals of the microtubule-based motor proteins ncd (non-claret disjunctional protein from Drosophila melanogaster) and kinesin. With ncd, the nucleotide b inding site was 70% occupied and the crystals were able to diffract X- rays to 2.5 Angstrom. The iodo-analog provided a useful isomorphous de rivative with overall phasing power 1.89 in the range of 25.0-2.5 Angs trom. With kinesin, 2'-IATP co-crystallized with the protein. The crys tals diffracted to at least 2.8 Angstrom with a phasing power of 1.73 in the range of 20.0-5.0 Angstrom. The analog was also found to be a s ubstrate for all of the enzymes tested, including creatine kinase, pyr uvate kinase, hexokinase, and myosin, with values of K-m and V-max tha t were within a factor of 10 of those for ATP. The analog supported mu scle contraction, relaxing fibers, and producing active tension with v alues not statistically different from those obtained with ATP. These results all suggest that this analog should be useful for providing a heavy-atom derivative for crystals of enzymes that bind ATP.