A number of health problems, such as muscle and metabolic diseases, are related to the role of ATP-utilizing enzymes which play a key role in a variety of biochemical pathways. All these enzymes require Mg(II) as an obligatory component of their reactions. Understanding the catalytic activity of the enzymes on a molecular basis requires knowledge of the structures of the reactants and products bound at the active sites of the enzymes. The goal of this project is to establish the active-site structures of three classes of ATP-utilizing enzymes viz. phosphoryl transfer, nucleotidyl transfer and pyrophosphoryl transfer by using NMR spectroscopy which is one of the few techniques available for structural investigations in liquids. Two types of experiments will be performed to obtain structural data: (i) spin-lattice relaxation measurements of 31P and selectively labelled 13C and 15N nuclei of the substrates in their enzyme-bound complexes in the presence of such activating paramagnetic cations as Mn(II) and Co(II) substituted for the normal activator Mg(II) and (ii) homonuclear proton NOE measurements on nucleotides in their enzyme complexes. The first method yields distances from the cation to the 31P, 13C, and 15N nuclei in the substrates and the second method provides information on the interproton distances on the nucleotides. Measurements will also be made using thionucleotide analogs, in place of normal nucleotides, in order to introduce stereochemical information into the structure data. The proposed measurements represent the maximal distance data that NMR methodology can provide on the substrates bound to the enzymes chosen. A special effort will be made to assess an acceptable range for each distance obtained from these studies as implied by the measurements themselves and the theoretical basis of calculating these distances from the data. Active-site structures compatible with the distance data acquired as above, will be constructed using appropriate iterative procedures.
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