th  - 1987 - 51st ENC Conference

WFI2
DETERMINATION OF ENZYME-STEROID BINDING SITES
BY CPMAS 13C NMRAND SELECTIVE EXCITATION
Vincent Bork* and Jacob Schaefer
Department of Chemistry, Washington University
St. Louis, MO 63130
Richard J. Auchus and Douglas F. Covey
Department of Pharmacology, Washington University
School of Medicine, St. Louis, MO 63110
The spatial proximity of inequivalent 13C nuclei in solids,
which have been multiply labeled with 13C, can be obtained from
spin transfer rates in CPMAS experiments involving selective
excitation. The selective excitation is used to invert just one
line in the spectrum which arises from a particular 13C label.
By varying a dipolar evolution time, 13C-13C connectivity is
revealed by new peaks in the spectrum obtained as a difference
between the normal CPMAS spectrum, and the CPMAS spectrum with
selective excitation. Confusing natural-abundance background
peaks disappear in the difference spectrum. This difference
technique can be used to identify 13C-labeled chemical bonds in
complex biological solids. For example, the technique has
demonstrated that the insoluble adduct between a [13C2]-
acetylenic steroid and human estradioldehydrogenase involves
binding of the drug to both lysine and cystine residues.