Quantitative measurement of the error in the cryptic stereospecificity of methylmalonyl‐CoA mutase

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Abstract
1. Samples of methylmalonyl coenzyme A and (2H3)methylmalonyl-CoA were prepared by a combination of chemical and enzymic methods. After ion-exchange chromatography the unlabeled methylmalonyl coenzyme A was pure, the deuterated substance contained 11-12% dephospho coenzyme A derivative. 2. The sample of unlabeled methylmalonyl coenzyme A was incubated in deuterated buffer with catalytic amounts of methylmalonyl coenzyme A mutase, epimerase, and coenzyme B12. The progress of the reaction was monitored directly by 1H-NMR spectroscopy at 500 MHz. After equilibrium was established, a slow mutase-catalyzed deuterium incorporation into migratable positions of succinyl coenzyme A was observed. 3. The sample of (2H3)methylmalonyl coenzyme A was incubated in unlabeled buffer with a mixture of methylmalonyl coenzyme A mutase, epimerase and coenzyme B12. In withdrawn aliquots, the reaction was interrupted by acidification and the lyophilized samples were examined by 1H-NMR spectroscopy in deuterium oxide. Both rearrangement and protium incorporation into migratable positions of succinyl coenzyme A were monitored. 4. At compoarable methylmalonyl coenzyme A to succinyl coenzyme A conversion rates, deuterium loss from migratable positions was 4-6 times faster than the corresponding protium loss. It is confirmed that the stereochemical error of the mutase is amplified by isotope discrimination when deuterium is in migratable positions, whereas it is diminished when protium is in migratable positions.