Chemistry of the inactivation of 4-aminobutyrate aminotransferase by the antiepileptic drug vigabatrin.
The chemical modification of pig liver 4-aminobutyrate aminotransferase by the antiepileptic drug 4-aminohex-5-enoate (Vigabatrin) has been studied. After inactivation by 14C-labeled Vigabatrin, the enzyme was digested with trypsin, and automated Edman degradation of the purified labeled peptide gave the sequence FWAHEHWGLDDPADVMTFSKK. Chymotryptic digestion of the tryptic peptide and sequencing of a resulting tripeptide identified the penultimate lysine residue of this peptide as the site of covalent modification. This lysine normally binds the coenzyme. Absorption spectroscopy demonstrated the absence of coenzyme from the tryptic peptide, and mass spectrometry showed its mass/charge ratio to be increased by 128. All of the bound coenzyme released after denaturation of the inactivated enzyme was as pyridoxamine phosphate. The structural nature of the modification is deduced, and mechanisms for its occurrence identified. Initially, 1 mol of radiolabeled inhibitor was bound per mol of monomer of the enzyme, although approximately half was released during denaturation and digestion, while the remainder was irreversibly bound. Coenzyme not released as pyridoxamine phosphate retained the absorbance characteristics of the aldimine, although the enzyme was completely inactive. Mass spectrometry of the sample of purified radiolabeled tryptic peptide revealed the presence of an approximately equal amount of a second fragment that contained no modification and from which the second lysine was absent, indicating that at the time of proteolysis the active site lysine was unaltered in 50% of the enzyme molecules.
Department of Biochemistry, University of Wales, Cardiff, Wales, United Kingdom.
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D000612:4-Aminobutyrate Transaminase / Q000037:antagonists & inhibitors*; D000595:Amino Acid Sequence; D000614:Aminocaproates / Q000494:pharmacology*; D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D013056:Spectrophotometry, Ultraviolet; D013552:Swine; D014357:Trypsin; D020888:Vigabatrin
D000614:Aminocaproates; D000927:Anticonvulsants; D000612:4-Aminobutyrate Transaminase; D014357:Trypsin; D020888:Vigabatrin