Further insights into the mechanism of action of methylmalonyl-CoA mutase by electron paramagnetic resonance studies.
Novel analogues of methylmalonyl-CoA and succinyl-CoA have been prepared and used for mechanistic investigations on the coenzyme-B12-dependent methylmalonyl-CoA mutase. 1-Carboxyethyl-CoA (1) and 2-carboxyethyl-CoA (2) as well as their sulphoxides (3 and 4) were moderately good inhibitors with Ki values 4-20 times higher than the Km for succinyl-CoA. 2-Carboxyethyl-CoA (2) and its sulphoxide 4 induced EPR signals when bound to the enzyme-coenzyme-B12 complex. The EPR spectrum of 2 and its sulphoxide 4 differed very much from those induced by the other substrates. In the case of 2 the EPR spectrum of the holoenzyme/inhibitor complex showed the presence of an organic radical coupled to cobal(II)amin. The same experiment with 4 leads to the formation of enzyme-bound cobal(II)amin with no detectable organic radical. The analogues 1 and 3 exhibited higher Ki values and did not induce EPR signals binding to the enzyme-coenzyme-B12 complex. Formyl-CoA and acrylate inhibited the enzyme synergistically but were unable to induce EPR signals and to form the product. Ethylmalonyl-CoA, known as a poor substrate, induced a similar but less intense EPR signal than the natural substrate methylmalonyl-CoA. The results are discussed in terms of the mechanism of the methylmalonyl-CoA mutase reaction.
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Lehrstuhl für Biochemie, Universität Karlsruhe, Germany.
Illich
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V
Rétey
J
J
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000494:pharmacology; D003038:Cobamides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008765:Methylmalonyl-CoA Mutase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D011424:Propionibacterium / Q000201:enzymology; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D003038:Cobamides; D004791:Enzyme Inhibitors; C102829:ethylmalonyl-coenzyme A; D008765:Methylmalonyl-CoA Mutase; C000913:cobamamide