The synthetic substrate succinyl(carbadethia)-CoA generates cob(II)alamin on adenosylcobalamin-dependent methylmalonyl-CoA mutase.
Succinyl(carbadethia)-coenzyme A, a synthetic substrate for adenosylcobalamin-dependent methylmalonyl-CoA mutase, has been prepared by a simplified procedure. When recombinant mutase was mixed with the synthetic substrate, the u.v./visible absorption spectrum of the bound cofactor changed rapidly to resemble that of cob(II)alamin, with an absorption maximum at 467 nm. Addition of the natural substrates, in contrast, produced only minor changes in the u.v./visible spectrum. The recent report of the generation of a complex e.p.r. spectrum on addition of substrate to the holo-methylmalonyl-CoA mutase was confirmed with the recombinant enzyme. The signals observed were stronger when the succinyl(carbadethia) analogue was used. Cobalt K-edge X-ray absorption spectroscopy confirmed that the addition of this analogue to holoenzyme leads to the generation of a cob(II)alamin-like species. These results strongly support the generation of cob(II)alamin during the 1,2-skeletal rearrangement catalysed by methylmalonyl-CoA mutase, as required if this enzyme follows the reaction pathway involving radical intermediates previously proposed for other adenosylcobalamin-dependent enzymes.
Keep
N H
NH
Department of Biochemistry, University of Cambridge, U.K.
Smith
G A
GA
Evans
M C
MC
Diakun
G P
GP
Leadlay
P F
PF
D000214:Acyl Coenzyme A / Q000138:chemical synthesis / Q000378:metabolism*; D003035:Cobalt / Q000378:metabolism; D003038:Cobamides / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D008765:Methylmalonyl-CoA Mutase / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013057:Spectrum Analysis; D013379:Substrate Specificity; D014805:Vitamin B 12 / Q000378:metabolism*
D000214:Acyl Coenzyme A; D003038:Cobamides; C083987:succinyl(carbadethia)-coenzyme A; D003035:Cobalt; D008765:Methylmalonyl-CoA Mutase; C000913:cobamamide; D014805:Vitamin B 12