Two-dimensional hyperfine sublevel correlation spectroscopy applied in thestudy of a Cu2+-[2-(alpha-hydroxyethyl)thiamin pyrophosphate]-[pentapeptide] system as a model of thiamin-dependent enzymes

To obtain structural information on the active-site of thiamin-dependent en zymes in solution, the ternary Cu2+-[Asp-Asp-Asn-Lys-Ile]-[2-(alpha -hydrox yethyl)thiamin pyrophosphate (HETPP)] system has been synthesized and studi ed by pulsed EPR (ESEEM and HYSCORE) spectroscopy in aqueous solution at ph ysiological pH. HYSCORE proved to be especially useful in elucidating the c oordination environment of the Cull ion. The present data show that, in the ternary Cu2+-[pentapeptide]-[HETPP] system at physiological pH, the peptid e backbone offers three coordination sites to the metal ion and the coordin ation sphere is completed by two additional phosphate oxygens and the nitro gen N(1 ') of the thiamin coenzyme. Thus the synthetic ternary system offer s the first example of a reliable structural model of the active site of th iamin-dependent enzymes in solution. The importance of our findings concern ing the N(1 ') coordination in the Cu2+-[HETPP]-[pentapeptide] system is di scussed in conjunction with the role of HETPP as an intermediate of thiamin catalysis.