WOODWARDS REAGENT-K INACTIVATION OF ESCHERICHIA-COLI L-THREONINE DEHYDROGENASE - INCREASED ABSORBENCY 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 inact ivated and develops a new absorbance peak at 347 nm when incubated wit h 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 similar to 50% as effecti ve. Tryptic digestion of WRK-modified TDH followed by HPLC fractionati on (pH 6.2) yields four 340-nm-absorbing peptides, two of which are ab sent from enzyme incubated with WRK and NAD(+). Peptide I has the sequ ence TAICGTDVH (TDH residues 35-43), whereas peptide II is TAICGTDVHIY (residues 35-45). Peptides not protected are TMLDTMNHGGR (III, residu es 248-258) and NCRGGRTHLCR (IV, residues 98-108). Absorbance spectra of these WRK-peptides were compared with WRK adducts of imidazole, 2-h ydroxy-ethanethiolate, and acetate. Peptides III and IV have pH-depend ent lambda(max) values (340-350 nm), consistent with histidine modific ation. Peptide I has a pH-independent lambda(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 affe ct enzyme activity. These results are the first definitive proof of WR K modifying cysteine and histidine residues of a protein and show that enzyme inactivation by WRK associated with the appearance of new abso rptivity at 340-350 nm does not establish modification of aspartate or glutamate residues, as has been assumed in numerous earlier reports.