STRUCTURAL CHARACTERIZATION OF THE ZINC SITE IN ESCHERICHIA-COLI L-THREONINE DEHYDROGENASE USING EXTENDED X-RAY-ABSORPTION FINE-STRUCTURE SPECTROSCOPY

Escherichia coli L-threonine dehydrogenase (TDH) is a homotetrameric p rotein which contains one Zn2+ ion per subunit and is a member of the medium chain, Zn2+-containing alcohol/polyol dehydrogenase family. TDH was subjected to extended X-ray absorption fine structure (EXAFS) spe ctroscopic analyses to explore what residues might bind the Zn2+; the EXAFS data are consistent with a tetrathiolate ligation sphere for the zinc atom. As a test of this proposed model, the oxidation state of t he six cysteine residues in the enzyme was evaluated. Under typical st orage conditions (4 degrees C with intermittent exposure to air; 50 mM Tris-HCl buffer, pH 8.4; 5 mM 2-mercaptoethanol, 2-ME) the TDH cystei ne residues undergo air-dependent oxidation to form one disulfide bond /subunit with no change in enzymatic activity. Measurements of the fre e thiol and disulfide levels during storage support this proposal. No disulfide bond forms in TDH when it is stored for up to 100 days at 4 degrees C under argon either with or without 5 mM 2-ME. The previously reported selective reactivity of Cys38 in native TDH towards either i odoacetate or Woodward's reagent K suggests that this residue is not i nvolved in disulfide bond formation. The EXAFS data reported here and the thiol/disulfide levels determined for TDH, together with the homol ogy of TDH to horse Liver alcohol dehydrogenase, suggest that the one Zn2+/subunit of TDH is non-catalytic and is probably bound by cysteine residues 93, 96, 99, and 107 in a structural zinc-binding loop of the protein. (C) 1993 Elsevier Science S.A. All rights reserved.