The secondary alpha-deuterium isotope effect at the site of the enzyme-catalyzed cis-trans isomerization of maleylacetone. Mechanistic implications

An equal mixture of monodeuterated isotopomers of maleylacetone, viz., 4-hy droxy-6-oxo-2,4-heptadienoic-2-d(1) acid (1) and 4-hydroxy-6-oxo-2,4-heptad ienoic-3-d(1) acid (2) was synthesized and subjected to catalyzed cia-trans isomerization by the maleylacetoacetate cia-trans isomerase-glutathione sy stem. These studies were designed to probe the detailed mechanism of enzyme -catalyzed cis-trans isomerization about the C-2-C-3 bond of the substrate. In the competitive reaction, 2 accumulates with respect to 1 at high exten t of reaction. H-1 NMR analysis indicates that 1 reacts 15% faster than 2 i n line with a nucleophilic addition mechanism at C-2 rather than a conjugat e addition mechanism at C-2 and C-3, to convert the C-2-C-3 double bond to a single bond to allow facile internal rotation.