Structure of the flavin adduct formed in the suicide reaction of alpha-hydroxybutynoate with D-lactate dehydrogenase.
The Zn-dependent flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii is irreversibly inactivated by the D form of the suicide substrate 2-hydroxy-3-butynoic acid. The process of inactivation involves formation of a new pink chromophore, which can be released in intact form from the protein and which was purified to homogeneity by affinity chromatography. Inactivation involves covalent addition of the suicide substrate to the flavin coenzyme. The optical spectra indicate an elongation of the flavin chromophore, and the chemical reactivity suggests a derivative of reduced flavin. The structure of this adduct was deduced from Fourier transform NMR, from the chemical properties, and from comparison with appropriate models, which were synthesized chemically. This structure involves the covalent linkage of the acetylenic inhibitor to positions N(5) and C(6) of the flavin coenzyme via carbon atoms 2 and 4 of the inhibitor to form an additional fused aromatic ring. The pink adduct can be reconverted to an isoalloxazine chromophore by reduction with borohydride and subsequent reoxidation with oxygen. This new isoalloxazine has the spectral properties of an isoflavin, and it is proposed to carry the moiety of the inactivator molecule as substituent at position C(6). The structure of the pink chromophore representing a cyclic adduct to the flavin positions N(5) and C(6) is compared to that of the adduct obtained from L-lactate oxidase from Mycobacterium smegmatis and the L form of the same inhibitor [C(4a)--N(5) cyclic adduct; Schonbrunn, A., Abeles, R. H., Walsh, C. T., Ghisla, S., Ogata, H., and Massey, V. (1976) Biochemistry 15, 1978]. This comparison allows deductions about the relative orientation of substrate, coenzyme, and active center functional groups in the two enzymes.[1]References
- Structure of the flavin adduct formed in the suicide reaction of alpha-hydroxybutynoate with D-lactate dehydrogenase. Ghisla, S., Olson, S.T., Massey, V., Lhoste, J.M. Biochemistry (1979) [Pubmed]
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