Complexation of Cu2+ by HETPP and the pentapeptide Asp-Asp-Asn-Lys-Ile: A structural model of the active site of thiamin-dependent enzymes in solution

To obtain structural information on the active site of thiamin-dependent en zymes in solution, we have studied the interactions of Cu2+ ions with 2-(al pha -hydroxyethyl)thiamin pyrophosphate (HETPP), the pentapeptide Asp-Asp-A sn-Lys-IIe surrounding the thiamin pyrophosphate moiety in the transketolas e enzyme, and the tertiary CU(2+)pentapeptide-HETPP system in aqueous solut ions at various pH values. In the binary Cu2+-pentapeptide system around ph ysiological pH, the bonding sites were the terminal NH2 group, the aspartat e beta -carboxylates, and a deprotonated peptide nitrogen, while, in the Cu 2+-HETPP system at the same pH, the Cu(II) was coordinated to the pyrophosp hate group and to the pyrimidine N(1') atom. It is found that, in the terti ary system at physiological pH, the, peptide bone offers three coordination sites to the metal ion, and the coordination sphere is completed by two ad ditional phosphate oxygens and the nitrogen N(1') of the thiamin coenzyme. The stability constants in the, tertiary system are higher than those in th e simpler CU2+-HETPP and CU2+-peptide systems. The present data show that t he coenzyme adopts the so-called S conformation in solution. The importance of our findings concerning the N(1') coordination and the S conformation i n the tertiary system is discussed in conjunction with the role of HETPP as an intermediate of thiamin catalysis.