An innovative technological and methodological approach will be developed to determine the three-dimensional active site structure of true intermediates of enzyme catalyzed reactions by application of a new method of electron nuclear double resonance (ENDOR) spectroscopy known as stochastic-ENDOR. The bacterial elongation factor Tu (EF-Tu) will be isolated as a perdeuterated protein with natural abundance 1H- or 19F- substituted amino acids selectively incorporated in active site positions through growth of auxotrophic strains of E. coli on perdeuterated medium. ENDOR spectra will be obtained for the EF-Tu.V02+(guanosine 5'-(5'-(beta, gamma-imido) triphosphate complex and the EF-TuV02+GTP complex (generated by photolytic cleavage of """"""""caged""""""""-GTP bound to the protein), in which the VO2+ ion serves as a paramagnetic substitute for Hg2+. Stochastic-ENDOR will be applied to determine whether this new methodology with greatly enhanced signal-to-noise capability over that of conventional, continuous wave (cw) END0R can be employed to detect the 1H and 19F resonances of residues site specifically incorporated into the deuterated protein. The limitations of application of stochastic-E&D0R will be assessed to determine the level of isotope enrichment necessary for accurate signal detection and to determine the resolving power of this new ENDOR methodology for partially overlapping resonances of residues distant (6-Il A) from the paramagnetic site. Determination of the principal hyperfine coupling components of specific active site residues will permit direct assignment of their distance from the metal ion and their orientation with respect to magnetic axes of the vo2+ ion. Successful application of stochastic-END0R will help to develop a new general method to determine the three-dimensional active site structure of enzyme reaction intermediates in solution.
