STEREOCHEMICAL KINETIC ISOTOPE EFFECTS FOR N RIBOHYDROLASES & TRANSFERASES

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The chemical bond that joins purines and pyrimidines to the anomeric carbon of ribose is susceptible to hydrolysis and phosphorolysis. The transition state structures for these reactions have been investigated by kinetic isotope effects in which isotopic labels are incorporated at every position where bonding changes are expected to occur at the transition state. The kinetic isotope effects provide information on the extent of bonding change at each atom in the transition state. By measuring isotope effects for 3H at the 1', 2', 4' and 5' positions, the 14C isotope effect at C1', the 15N isotope effect at N9, and 18O isotope effect at the 4'O of nucleosides, it is possible to construct a complete structure for the transition state. Comparison of the chemical solvolysis of the N-ribosidic bond with the same reactions catalyzed by enzymatic N-riboyhydrolases, makes it possible to establish the novel contributions made by the enzyme to transition state stabilization. While this approach is thorough and far-reaching, a major barrier to implementation is the difficulty of obtaining specifically labeled substrates. The National Tritium Labelling Facility is essential for the conduct of this research. Three of the tritium labels which are essential for the transition state analysis are provided through the efforts of the Tritium Resource. TITLE: Stereochemical Kinetic Isotope Effects for N-Ribohydrolases and Transferases (Continued) Several enzymatic systems are under investigation in the Schramm laboratory. All these projects include investigation of fundamental principles of catalysis and applications of transition state information to the design of enzymatic transition state inhibitors. The enzymatic systems currently under investigation include: Purine N-ribohydrolases from protozoan parasites: The organisms are purine auxotrophs and use a novel family of N-ribohydrolases to salvage exogenous purines. The enzymes are characterized by transition state structures with ribooxocarbenium character achieved by activation of the leaving group or by activation of the ribosyl to reach similar transition states. Knowledge of the transition state structures has permitted the design and synthesis of powerful transition state inhibitors which are specific for the isozymes found in different protozoa. These enzymes are not found in mammals, making them suitable targets for inhibitor design. Ribose 5-phosphate is labeled from 3H20 at the NTLF for synthesis of labeled nucleosides, and stereospecific 2'-pro-R and 2'-pro-S tritium nucleosides are synthesized from this material or chemically by NTLF scientists. Stereospecific isotope effects provide accurate geometry information at the transition state, and there is no other way to extract this information. Purine nucleoside phosphorylase (PNP) of human origin: The genetic deficiency of human PNP causes T-cell deficiency. T-cell responses cause type IV autoimmune disorders and transition state inhibitors have promise in treatments of tissue transplant rejection, psoriasis, rheumatoid arthritis, inflammatory bowel disease and many related immune disorders. The transition state has been solved using the tritiated substrates prepared at the NTLF. CD38 is a human enzyme which synthesizes cyclic-ADP ribose: This newly discovered molecule is thought to be synthesized as a signalling molecule which causes the release of intracellular calcium. Efforts are underway to measure the kinetic isotope effects from this enzyme. To accomplish this goal, tritium labeled ribose 5-phosphate prepared at the NTLF is used to synthesize 2'-3H NAD+ for measurement of isotope effects. Ricin A-chain from castor beans: Ricin A-chain is one of the most powerful cytotoxins, entry of a single molecule into a mammalian cell is lethal. The toxin is in clinical trials as an immunoconjugate to treat otherwise incurable cancers. Transition state analysis of the RNA N-ribohydrolase activity of ricin A-chain is proposed to lead to powerful inhibitors of the toxin which can be used as rescue agents in ricin immunotherapy to decrease the side effects of this therapy. RNA and DNA stem-loop structures are prepared from tritium labeled nucleotides prepared from stereospecifically labeled adenine nucleotides labeled at the NTLF. All of the precursors for two of the isotope effects are synthesized at NTLF. Future Projects: In collaboration with the staff at NTLF we propose to stereospecifically label the pro-R and pro-S hydrogens at the 5'C of nucleosides and nucleotides. This seemingly remote position has been found to give surprisingly large kinetic isotope effects which arise from distortion of the sp3 geometry of the 5'C at the transition state. These measurements have never been made in any enzymatic system to our knowledge. We look forward to continuing these leading-edge isotope effect measurements, which are completely dependent on the availability of the NTLF.