NEW CHEMICAL APPROACHES TO ADP-RIBOSYL TRANSFER

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ADP-ribosyl transferases and the related NAD glycohydrolases are a series of enzymes which catalyze the transfer of the adenosine diphosphate ribose portion of NAD to a variety of nucleophilic acceptors or to water. The biological significance of this variety of posttranslational modifications is not yet clear, although several important regulatory roles have been suggested. A proposal is advanced herein for the design of a series of compounds which are predicted to function as "suicide" enzyme inactivators (i.e., as mechanism based irreversible enzyme inhibitors) for NAD glycohydrolases and related ADP-ribosyl transferases. The mechanism of inhibition exploits the well documented pyridine base exchange activity of the NAD glycohydrolases. An effort first will be made to obtain the requisite compounds and to determine whether they are indeed "suicide" inactivators for enzymes known to carry out the pyridine base exchange reaction. Next, an attempt will be made to extend this type of inhibition to various ADP-ribosyl transferases. Such inhibitors will be useful in labeling and identifying active site residues in susceptible enzymes. Once the target enzymes have been identified, the inhibitors will be used to probe the physiological function(s) of the targets in cultured cells. Another strategy for inhibitor design is advanced, this one involving the production of a "non-cleavable" NAD analog which is predicted to be a non-covalent inhibitor of ADP-ribosyl transferases. Such a compound would be valuable for in vitro studies on ADP-ribosyl transferases, particularly the poly(ADP-ribose) synthetase isolated from mammalian nuclei. Other mechanistic experiments on this enzyme are proposed. These begin with the development of a sensitive continuous assay and a subsequent careful kinetic study designed to detect any intermediates in the reaction.