Quantitative measurement of the error in the cryptic stereospecificity of methylmalonyl-CoA mutase.
1. Samples of methylmalonyl-CoA and (2H3)methylmalonyl-CoA were prepared by a combination of chemical and enzymic methods. After ion-exchange chromatography the unlabelled methylmalonyl-CoA was pure, the deuterated substance contained 11-12% dephospho-CoA derivative. 2. The sample of unlabelled methylmalonyl-CoA was incubated in deuterated buffer with catalytic amounts of methylmalonyl-CoA 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-catalysed deuterium incorporation into migratable positions of succinyl-CoA was observed. 3. The sample of (2H3)methylmalonyl-CoA was incubated in unlabelled buffer with a mixture of methylmalonyl-CoA mutase, epimerase and coenzyme B12. In withdrawn aliquots, the reaction was interrupted by acidification and the lyophilised samples were examined by 1H-NMR spectroscopy in deuterium oxide. Both rearrangement and protium incorporation into migratable positions of succinyl-CoA were monitored. 4. At comparable methylmalonyl-CoA to succinyl-CoA 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.
D000214:Acyl Coenzyme A / Q000138:chemical synthesis / Q000378:metabolism; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003903:Deuterium / Q000378:metabolism; D004735:Energy Transfer; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008765:Methylmalonyl-CoA Mutase / Q000378:metabolism*; D011424:Propionibacterium / Q000201:enzymology; D012997:Solvents
D000214:Acyl Coenzyme A; D012997:Solvents; D003903:Deuterium; C012046:succinyl-coenzyme A; D007535:Isomerases; D008765:Methylmalonyl-CoA Mutase