Woodward's reagent K inactivation of Escherichia coli L‐threonine dehydrogenase: Increased absorbance at 340–350 nm is due to modification of cysteine and histidine residues, not aspartate or glutamate carboxyl groups

L‐Threonine dehydrogenase (TDH) from Escherichia coli is rapidly inactivated and develops a new absorbance peak at 347 nm when incubated with N ‐ethyl‐5‐phenylisoxazolium‐3′‐sulfonate (Woodward's reagent K, WRK). The cofactors, NAD + or NADH (1.5 mM), provide complete protection against inactivation; L‐threonine (60 mM) is ∼50% as effective. Tryptic digestion of WRK‐modified TDH followed by HPLC fractionation (pH 6.2) yields four 340‐nm‐absorbing peptides, two of which are absent from enzyme incubated with WRK and NAD + . Peptide I has the sequence TAICGTDVH (TDH residues 35–43), whereas peptide II is TAICGTDVHIY (residues 35–45). Peptides not protected are TMLDTMNHGGR (III, residues 248–258) and NCRGGRTHLCR (IV, residues 98–108). Absorbance spectra of these WRK‐peptides were compared with WRK adducts of imidazole, 2‐hydroxy‐ethanethiolate, and acetate. Peptides III and IV have pH‐dependent λ max values (340–350 nm), consistent with histidine modification. Peptide I has a pH ‐independent λ max (350 nm) indicating that a thiol is modified. WRK, therefore, does not react specifically with carboxyl groups in this enzyme, but rather modifies Cys‐38 in the active site of TDH; modification of His‐105 and His‐255 does not affect enzyme activity. These results are the first definitive proof of WRK modifying cysteine and histidine residues of a protein and show that enzyme inactivation by WRK associated with the appearance of new absorptivity at 340–350 nm does not establish modification of aspartate or glutamate residues, as has been assumed in numerous earlier reports.