The substrate-dependent homolysis of the cobalt-carbon bond and generation of organic radicals in the coenzyme-B, , -methylmalonyl-CoA-mutase complex have been demonstrated by EPR measurements. Both the natural substrate methylmalonyl-CoA, its '3C-substituted analogue and the nonhydrolysable synthetic substrates succinyl-dethia(carba)-CoA, succinyl-dethia(dicarba)-CoA and 4-carboxy-2-0x0-butyl-CoA induced similar but not identical EPR signals. 3-Carboxypropyl-CoA, a novel competitive inhibitor, has been synthesised. Its K, value of 89 2 6 pM was in the same range as the K,, of succinyl-CoA. Using [S'-3H]adenosylcobalamin, an enzyme-dependent tritium transfer to the inhibitor has been shown. The enzyme-coenzyme-inhibitor complex also exhibited EPR signals that were less structured and less intensive than the corresponding signals with active substrates.These results prove that the inhibitor also induces cobalt-carbon bond homolysis and undergoes reversible hydrogen transfer but not rearrangement.Methylmalonyl-CoA mutase, a coenzyme-B, ,-dependent enzyme, catalyses the rearrangement of (2R)-methylmalonylCoA to succinyl-CoA (Eqn 1). In analogy to other coenzyme-B,,-dependent reactions (for a review see [l]) and some chemical models [2, 31, it is expected that the first chemical step comprises homolysis of the cobalt-carbon bond and the so-generated highly reactive 5'-adenosyl radical abstracts a hydrogen atom from the (non-activated) methyl group of methylmalonyl-CoA. Thus, during the rearrangement, several organic radical species and the paramagnetic cob(I1)alamine are postulated as intermediates (Scheme 1). analogues, succinyl-dethia(carba)-coenzyme A (succinyl-CH,-CoA) 2, were published in 1986 [6]. A publication with full experimental details of the synthesis, as well as the spectral and kinetic characterisation of succinyl-CH,-CoA 2, succinyl-CH,CH,-CoA 3 and 4-carboxy-2-0x0-butyl-CoA 4 appeared recently [7]. Using our experimental procedure [6, 8, 91, Leadlay and coworkers [lo] also published the synthesis of 2 and its ability to induce EPR signals in the methylmalonyl-CoA-mutase -coenzyme-B , , complex. Here we describe in detail the EPR signals obtained with substrates, non-hydrolysable substrate analogues and an inhibitor of the methylmalonyl-CoA mutase. In addition, the synthesis and behaviour of the novel inhibitor 3-carboxypropyl-CoA 5 is also described. EXPERIMENTAL PROCEDURES EquipmentAll EPR measurements were recorded with an ESP 300 E 10112 ESR spectrometer (Bruker Analytische MeBtechnik) at a microwave power of 5 mW. An NMR gaussmeter and a frequency counter (model HP 5350 B) were used to calibrate the magnetic field. A cylindrical TM resonator served as sample head. The EPR spectra were recorded at 77 K. NMR spectra were produced using Bruker WH 250 and AM 400 spectrometers. The thin-layer scanner was obtained from Fa. Berthold (Germany). For all other experiments, standard laboratory equipments were used. MaterialsCoenzyme B,, (adenosylcobalamin), KH,PO,, K,HPO,, y-butyrolactone, LiC1, NaOH, pe...

Only after the addition of the substrate, methylmalonyl‐CoA, to the binary complex of highly purified methylmalonyl‐CoA mutase and coenzyme B12 is a clear ESR signal observed. This proves that radical intermediates that have only been postulated in the past are indeed involved in the enzymatic rearrangement of the carbon skeleton.

Erst nach Zugabe des Substrats Methylmalonyl‐CoA zum binären Komplex aus hochgereinigter Methylmalonyl‐Co‐A‐Mutase und Coenzym B12 tritt ein deutliches ESR‐Signal auf. Dies beweist, daß während der enzymatischen Umlagereung des Kohlenstoffgerüsts radikalische Zwischenstufen durchlaufen werden, die bei analogen Umwandlungen bisher nur postuliert werden.

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Nicht als Redoxreagens, sondern als Elektrophil fungiert NAD+ im zweiten Schritt des Histidin‐Abbaus. Bei dieser bisher einmaligen enzymatischen Reaktion wird Wasser an Urocaninsäure 1 (unter Bildung von 2) addiert. Der vorgeschlagene Mechanismus wurde nun NMR‐spektroskopisch anhand des 13C‐markierten Addukts 3 erhärtet. R = ADP‐Ribosyl, 0 = 13C.