Abstract
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 ATP 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 binding site was 70% occupied and the crystals were able to diffract X-rays to 2.5 A. The iodo-analog provided a useful isomorphous derivative with overall phasing power 1.89 in the range of 25.0-2.5 A. With kinesin, 2'-IATP co-crystallized with the protein. The crystals diffracted to at least 2.8 A with a phasing power of 1.73 in the range of 20.0-5.0 A. The analog was also found to be a substrate for all of the enzymes tested, including creatine kinase, pyruvate kinase, hexokinase, and myosin, with values of Km and Vmax that were within a factor of 10 of those for ATP. The analog supported muscle contraction, relaxing fibers, and producing active tension with values 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.
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- Alessi D. R., Corrie J. E., Fajer P. G., Ferenczi M. A., Thomas D. D., Trayer I. P., Trentham D. R. Synthesis and properties of a conformationally restricted spin-labeled analog of ATP and its interaction with myosin and skeletal muscle. Biochemistry. 1992 Sep 1;31(34):8043–8054. doi: 10.1021/bi00149a039. [DOI] [PubMed] [Google Scholar]
- Cooke R., Franks K., Luciani G. B., Pate E. The inhibition of rabbit skeletal muscle contraction by hydrogen ions and phosphate. J Physiol. 1988 Jan;395:77–97. doi: 10.1113/jphysiol.1988.sp016909. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dale R. M., Livingston D. C., Ward D. C. The synthesis and enzymatic polymerization of nucleotides containing mercury: potential tools for nucleic acid sequencing and structural analysis. Proc Natl Acad Sci U S A. 1973 Aug;70(8):2238–2242. doi: 10.1073/pnas.70.8.2238. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grossbard L., Schimke R. T. Multiple hexokinases of rat tissues. Purification and comparison of soluble forms. J Biol Chem. 1966 Aug 10;241(15):3546–3560. [PubMed] [Google Scholar]
- Hai T. T., Abo M., Hampton A. Species- or isozyme-specific enzyme inhibitors. 9. Selective effects in inhibitions of rat pyruvate kinase isozymes by adenosine 5'-diphosphate derivatives. J Med Chem. 1982 Oct;25(10):1184–1188. doi: 10.1021/jm00352a017. [DOI] [PubMed] [Google Scholar]
- Hohnadel David C., Cooper Cecil. The effect of structural alterations on the reactivity of the nucleotide substrate of rabbit muscle pyruvate kinase. FEBS Lett. 1973 Feb 15;30(1):18–20. doi: 10.1016/0014-5793(73)80609-x. [DOI] [PubMed] [Google Scholar]
- Pate E., Nakamaye K. L., Franks-Skiba K., Yount R. G., Cooke R. Mechanics of glycerinated muscle fibers using nonnucleoside triphosphate substrates. Biophys J. 1991 Mar;59(3):598–605. doi: 10.1016/S0006-3495(91)82275-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sablin E. P., Fletterick R. J. Crystallization and preliminary structural studies of the ncd motor domain. Proteins. 1995 Jan;21(1):68–69. doi: 10.1002/prot.340210108. [DOI] [PubMed] [Google Scholar]
- Wang D., Pate E., Cooke R., Yount R. Synthesis of non-nucleotide ATP analogues and characterization of their chemomechanical interaction with muscle fibres. J Muscle Res Cell Motil. 1993 Oct;14(5):484–497. doi: 10.1007/BF00297211. [DOI] [PubMed] [Google Scholar]
- Yount R. G. ATP analogs. Adv Enzymol Relat Areas Mol Biol. 1975;43:1–56. doi: 10.1002/9780470122884.ch1. [DOI] [PubMed] [Google Scholar]