NMR STUDIES OF ENZYME MECHANISMS AND METABOLIC PATHWAYS

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This proposal has the following broad long-term objectives: (1) Development of novel NMR experiments to study enzymes in action and to unravel the structural details of drug-receptor interaction. (2) Understanding the detailed mechanism of the biosynthetic enzymes of several important, complex metabolic pathways leading to (a) penicillin, (b) lanosterol and (c) heme, using high field NMR of regiospecifically 13C enriched substrates and purified enzymes obtained by cloning and overexpression. (3) The study of covalent binding of mechanism-based inhibitors of enzymes which play a key role in the diseases of emphysema (elastase), AIDS (HIV-1 protease) and acute intermittent porphyria (ALA synthase, porphobilinogen deaminase, uro'gen III synthase). (4) The conformations and mode of binding in solution and solid state of the anti-tumor drugs vinblastine (VLB) and taxol to their receptors in tubulin. (5) In vivo NMR studies of intracellular metabolism. The experimental methods will combine high field NMR spectroscopy in solution (300, 500 MHz) and in the solid state (300 MHz) with regio- and stereo-specific synthesis of 13C enriched substrates and inhibitors using direct and indirect (INEPT, INADEQUATE, NOESY) spectroscopy with inverse (1H/13C) and wide bore (20 mm) probes in conjunction with cryoenzymology of the requisite enzymes obtained by cloning, overexpression and purification at greater than or equal to 1.0 millimolar concentrations necessary for the detection of enzyme-bound species. The significance of the approach, which uniquely combines organic synthesis, high field NMR, low temperature (cryo) enzymology and molecular biology, is that active sites and drug-receptor interactions can be accurately mapped via isotopic enrichment of the binding small molecule and, most importantly, the time course of the biochemical events can be successfully monitored at the structural level by low temperature NMR spectroscopy.