Structure of the flavin adduct formed in the suicide reaction of .alpha.-hydroxybutynoate with D-lactate dehydrogenase

Abstract

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 in-

Studies on glycollate oxidase from pea leaves determination of stereospecificity and mode of inhibition by α-hydroxybutynoate

“…For the P. sativum enzyme no details were given on the mechanism of inactivation or the nature of the modification [12]. a-Hydroxybutynoic acid has been shown to in-'\ activate L-lactate oxidase and D-Iactate dehydro-,J genase, among other a-hydroxy acid oxidases [14][15][16][17], via formation of covalent flavin adducts. The structures of the inactivation products were shown to reflect the L-or D-specificity of the enzyme.…”

Suicide inactivation of the flavoenzyme D-lactate dehydrogenase by .alpha.-hydroxybutynoate

“…From its oxygen reactivity, this intermediate must have a structure related to or identical with 1,5-dihydroflavin; i.e., a substitution at positions C(4a) or N(5) similar to that found with other known acetylenic inhibitors cannot have occurred. As discussed in the following paper, this intermediate from mechanistic considerations could be unique to the carbanion pathway (Ghisla et al, 1979).…”

“…Those a-hydroxy acid oxidizing flavoenzymes for which spectral data have been published all appear to form adducts resembling that found with lactate oxidase (Lederer, 1974;Cromartie & Walsh, 1975). The structural elucidation of the new adduct is detailed in the following paper (Ghisla et al, 1979).…”

“…The following paper (Ghisla et al, 1979) focuses on the elucidation of the structure of the modified flavocoenzyme.…”

“…Maximal pink flavin formation was complete by the time the spectrum was recorded. The relevance of this intermediate to the two possible inactivation mechanisms is discussed more thoroughly in the following paper (Ghisla et al, 1979) in terms of chemical structure. It should suffice to say, however, at this point that this intermediate from chemical considerations is most likely to have arisen from the carbanion inactivation pathway, lending further support to a carbanion mechanism for this enzyme.…”