THE SYNTHETIC SUBSTRATE SUCCINYL(CARBADETHIA)-COA GENERATES COB(II)ALAMIN ON ADENOSYLCOBALAMIN-DEPENDENT METHYLMALONYL-COA MUTASE

Succinyl(carbadethia)-coenzyme A, a synthetic substrate for adenosylco balamin-dependent methylmalonyl-CoA mutase, has been prepared by a sim plified procedure. When recombinant mutase was mixed with the syntheti c substrate, the u.v./visible absorption spectrum of the bound cofacto r changed rapidly to resemble that of cob(II)alamin. with an absorptio n maximum at 467 nm. Addition of the natural substrates. in contrast. produced only minor changes in the u.v./visible spectrum. The recent r eport of the generation of a complex e.p.r. spectrum on addition of su bstrate to the holo-methylmalonyl-CoA mutase was confirmed with the re combinant enzyme. The signals observed were stronger when the succinyl (carbadethia) analogue was used. Cobalt K-edge X-ray absorption spectr oscopy confirmed that the addition of this analogue to holoenzyme lead s to the generation of a cob(II)alamin-like species. These results str ongly support the generation of cob(II)alamin during the 1,2-skeletal rearrangement catalysed by methylmalonyl-CoA mutase, as required if th is enzyme follows the reaction pathway involving radical intermediates previously proposed for other adenosyl-cobalamin-dependent enzymes.