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 approximately 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-hydroxyethanethiolate, and acetate. Peptides III and IV have pH-dependent lambda max values (340-350 nm), consistent with histidine modification. Peptide I has 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 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.
Johnson
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Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606, USA.
Dekker
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D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors*; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000187:drug effects; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors*; D003545:Cysteine / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D018698:Glutamic Acid / Q000187:drug effects; D006639:Histidine / Q000187:drug effects*; D007555:Isoxazoles / Q000494:pharmacology*; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D012997:Solvents / Q000494:pharmacology*; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000032:analysis
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D007555:Isoxazoles; D012997:Solvents; D013438:Sulfhydryl Compounds; D001224:Aspartic Acid; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D018698:Glutamic Acid; D006639:Histidine; D000429:Alcohol Oxidoreductases; C023159:L-threonine 3-dehydrogenase; D003545:Cysteine