Structure of the Product of the Enzymatic Reaction between Maleimides and Substituted 4-Carbon Amino Acids

• Published in: The Journal of biological chemistry Vol. 241; no. 14; pp. 3340 - 3346
• Main Authors: Flavin, Martin, Tsunakawa, Sukenari
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 25.07.1966
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)96468-9

In elimination reactions, catalyzed by pyridoxal phosphate enzymes, from β- or γ-substituted 4-carbon amino acids, a transient intermediate is trapped by N -ethylmaleimide, leading to the accumulation of a new product in place of α-ketobutyrate. From examination of degradation products, this compound has tentatively been assigned the structure α-keto-3-[3'-( N '-ethyl-2',5'-dioxopyrrolidyl)]butyric acid (KEDB). KEDB has now been isolated in pure form as its dinitrophenylhydrazone (DNPH) derivative, as has the corresponding compound, α-keto-3-[3'-(2',5'-dioxopyrrolidyl)]butyric acid (KDB), formed when maleimide was substituted for N -ethylmaleimide. The molar specific radioactivity (compared with that of a labeled precursor) and ultraviolet absorbance of KEDB-DNPH were compatible with the molecular weight, 407, of the proposed structure, and the infrared spectrum indicated the presence of an intact succinimide ring. The presence or absence of optical activity could not be determined because of rapid racemization, as shown by interconversion of diastereoisomers, during the isolation procedure. The elementary composition of all the ions in a mass spectrum of KEDB-DNPH was determined. The peak of highest mass corresponded to the proposed structure less CO 2 . However, the spectrum of α-ketobutyrate-DNPH also showed only a weak molecular ion, and an intense peak reflecting decarboxylation. The spectrum of KDB-DNPH served to distinguish fragments originating from DNPH, which were of the same mass as those from KEDB-DNPH, from those containing the succinimidyl group, which were 28 mass units (C 2 H 4 ) lower. The elemental composition of fragments thus shown to be derived from the succinimidyl moiety (C 10 H 13 NO 4 ) of KEDB-DNPH, and containing 9, 8, or 6 carbon atoms, was consistent with plausible fragmentations of a structure in which N -ethylsuccinimide is connected with the carbonyl carbon of the α-keto acid moiety by —(CH 3 )CH—, whereas the low abundance of 7-carbon fragments was evidence against a linkage through a —CH 2 CH 2 — bridge.