Further Insights into the Mechanism of Action of Methylmalonyl‐CoA Mutase by Electron Paramagnetic Resonance Studies

Abstract

Novel analogues of methylmalonyl-CoA and succinyl-CoA have been prepared and used for mechanistic investigations on the coenzyme-B ,,-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 K, values 4-20 times higher than the K, for succinyl-CoA. 2-Carboxyethyl-CoA (2) and its sulphoxide 4 induced EPR signals when bound to the enzyme-coenzyme-B,, 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 holoenzymelinhibitor complex showed the presence of an organic radical coupled to cobal(I1)amin. The same experiment with 9 leads to the formation of enzyme-bound cobal(I1)amin with no detectable organic radical. The analogues 1 and 3 exhibited higher K, values and did not induce EPR signals binding to the enzyme-coenzyme-B,, 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.Keywords: coenzyme B ; methylmalonyl-CoA analogs ; EPR spectra; inhibition kinetics.The coenzyme-B,,-dependent methylmalonyl-CoA mutase catalyses the rearrangement of (R)-methylmalonyl-CoA to succinyl-CoA (Eqn 1) [I, 21. As for other coenzyme-B,,-dependent enzymes catalysing carbon skeleton rearrangements, e.g. 2-methyleneglutarate mutase [3] and glutainate mutase [4], the catalytic reaction is initiated by homolysis of the Co-C bond of the coenzyme which occurs upon binding the substrate to the holoenzyme and results in the formation of a highly reactive 5'-deoxyadenosyl radical and the paramagnetic Coz+. The 5'-deoxyadenosyl radical abstracts a hydrogen atom from the (nonactivated) methyl group of methylmalonyl-CoA and the thus generated substrate radical rearranges to the product radical which abstracts the hydrogen atom back from the transiently formed 5'-deoxyadenosine. Thus, during the rearrangement, several organic radical species and the paramagnetic cob(I1)alamin are postulated as intermediates (Scheme 1). Recent EPR investigations using methylmalonyl-CoA [ 5 ] , its dethia(carba) derivatives and the inhibitor 3-carboxypropyl-CoA (61 as a substrate documented the formation of Co2+ in the frozen steady-state of the enzymic reaction. The characterisation of organic radicals however failed because the EPR signals were too complex. The X-ray structure of the coenzyme-B ,,-binding fragment of the methionine synthetase (71 revealed the replacement of the dimethylbenzimidazole of the coenzyme by a His residue of the protein. The co-ordination of a His to the Co2+ was then confirmed for glutamate mutase [ 81 and methylmalonyl-CoA mutase [9] by incorporating "N-labelled histidine into the enzymes followed by EPR measurements. Recently...

Proton Transfer from Histidine 244 May Facilitate the 1,2 Rearrangement Reaction in Coenzyme B12-dependent Methylmalonyl-CoA Mutase

“…The driving force for this conformational rotation is not clear. Furthermore, formyl-CoA and acrylate inhibit methylmalonyl-CoA mutase with a very high K i , which is Ͼ100-fold larger than the K m for succinyl-CoA (31 reassociation pathway requires significantly greater energy (93.2 kJ mol Ϫ1 versus 46.9 kJ mol Ϫ1 for direct rearrangement (18)). Thus, there are no compelling reasons at present to consider this mechanism for the methylmalonyl-CoA mutasecatalyzed reaction.…”

Alternative Pathways for Radical Dissipation in an Active Site Mutant of B12-Dependent Methylmalonyl-CoA Mutase

“…We tested two analogs with extended carbon skeletons: ethylmalonyl-CoA, a poor alternative substrate for wild-type enzyme (20,21), and allylmalonyl-CoA, which is predicted to stabilize the substrate allylic radical. With wild-type enzyme, both analogs act as competitive inhibitors and exhibit similar K i values (4 50 μM) ( Table I).…”

“…The kinetic parameters for inactivation by allylmalonyl-CoA were obtained by UV-visible stopped-flow spectrophotometry by monitoring the formation of cob(II)alamin and H 2 OCbl. Holo-Y243A (9 μM after mixing) was rapidly mixed with various concentrations of allylmalonyl-CoA (7,14,21,30, 50 and 100 μM after mixing) in 50 mM potassium phosphate buffer, pH 7.5 at 20°C. Cob(II)alamin formation was monitored by a decrease in absorbance at 525 nm over a 0.5 sec period and H 2 OCbl formation was monitored by an increase at 351 nm over an 8 sec period.…”

Synthesis of Enantiomerically‐Pure [13C]Aristeromycylcobalamin and Its Reactivity in Dioldehydratase, Glyceroldehydratase, Ethanolamine Ammonia‐Lyase and Methylmalonyl‐CoA Mutase Reactions

“…[12] Cob(i)alamin was then alkylated with the 6'-chloro-6'-deoxyaristeromycins (10) and (11) to afford aristeromycylcobalamin (12), and its 2',3'-bis-epi analogue (13) in 60 and 55 % yield, respectively (Scheme 3). Due to the high light sensitivity of the alkylcobalamins, both of the last reaction steps and the subsequent purification by HPLC was carried out in the dark.…”

“…The corresponding enzymes are able to accelerate the homolysis of the cobaltÀcarbon bond by a factor of up to 10 12 , [6,7] but only on substrate binding. [8±10] Obviously, a portion of the binding energy is used to induce a conformational change which weakens the cobaltÀcarbon bond.…”