K. Tanaka, D. Aberhart, J. Amster
Aug 25, 1980
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Journal
The Journal of biological chemistry
Abstract
To study the physiological role of keto-enol tautomerization at the 2-keto acid stage in branched chain amino acid metabolism, we administered valine and isobutyric acid, both chirally labeled with stable isotopes, to vitamin Blz-folate-deficient rats. The intramolecular isotope enrichment in the urinary methylmalonic acid from these rats was determined using gas chromatography-mass spectrometry and selected ion monitoring. Urinary methylmalonic acid from two rats loaded with (2RS,3S)-[4-13C]valine was labeled predominantly at one of the carboxyl carbon atoms. The 2-methyl group was not labeled to a significant degree. This indicates that in valine metabolism, isobutyryl-CoA dehydrogenation occurred stereospecifically by abstracting one hydrogen each from the (2proS)methyl and the a-methine groups of isobutyrate, confirming our previous results (Amster, J., and Tanaka, K. (1980) J. Biol. Chem 255, 119-120), and that keto-enol tautomerization did not occur to a significant degree under these conditions. Urinary methylmalonic acid excreted by a rat loaded with (2S,3S)-[4,4,4-’H3]valine was labeled 19 atom % with three ’H at the 2-methyl group. However, this ‘H3 enrichment in the 2-methyl group is greatly exaggerated by a selective retention of the 2-[’H3]methylmalonic acid due to the deuterium isotope effect on the residual methylmalonyl-CoA mutase. This was indicated by results on another rat loaded with (2S)-[3,3,3‘H3]isobutyric acid. It excreted a large amount of 3hydroxyisobutyric acid in addition to methylmalonic acid. Urinary methylmalonic acid from this rat was labeled 29 atom % with three ‘H at the 2-methyl group while only 5 atom 8 of urinary 3-hydroxyisobutyrate was labeled with three ‘H at the 2-methyl group. The remaining 95% of urinary 3-hydroxyisobutyrate was labeled with two ’H at the hydroxymethyl group.