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

Abstract

Methylmalonyl-CoA mutase catalyzes the adenosylcobalamin-dependent rearrangement of (2R)-methylmalonyl-CoA to succinyl-CoA. The crystal structure of the enzyme reveals that Y243 is in van der Waals contact with the methyl group of the substrate and suggests a possible role for it in the stereochemical control of the reaction. This hypothesis was tested by designing a molecular hole by replacing the phenolic side chain of Y243 with the methyl group of alanine. The Y243A mutation lowered the catalytic efficiency >4 × 10 4 -fold compared to wild type enzyme, the K Mapp for the cofactor ~4-fold, and the cob(II)alamin concentration under steady-state turnover conditions ~2-fold. However, the mutation did not appear to lead to loss of the stereochemical preference for the substrate. The Y243A mutation is expected to create a cavity and should in principle, allow accommodation of bulkier substrates. To test this, we used ethylmalonyl-CoA and allylmalonyl-CoA, as alternate substrates. Surprisingly, both analogs resulted in suicidal inactivation albeit in an O 2 -dependent and O 2 -independent fashion, respectively. The inactivation by allylmalonyl-CoA was further investigated and revealed formation of cob(II)alamin at a ~1.5-fold higher rate than with wild-type mutase under single turnover conditions. Product analysis revealed a stoichiometric mixture of 5′-deoxyadenosine, aquocobalamin and allylmalonyl-CoA. Taken together, these results are consistent with an internal electron transfer from cob(II)alamin to the substrate analog radical. These studies serve to emphasize the fine control exerted by Y243 in the vicinity of the substrate to minimize radical extinction in side reactions.Methylmalonyl-CoA mutase catalyzes the reversible isomerization of methylmalonyl-CoA to succinyl-CoA and is dependent on 5′-deoxyadenosylcobalamin (AdoCbl) 1 or coenzyme B 12 for activity (Figure 1) (1). In this reaction, the AdoCbl cofactor is used as a radical reservoir that generates via homolysis of theCo-carbon bond, a pair of radicals: cob(II)alamin and a reactive 5′-deoxyadenosyl radical (Ado•). The latter abstracts a hydrogen atom from the substrate generating a substrate-centered radical that, in turn, rearranges to a product-centered radical. The remainder of the catalytic cycle is completed by reversal of the sequence of steps in the first half of the reaction (Figure 1).Two questions of long-standing interest regarding AdoCbl-dependent enzymes are how the inherent kinetic inertness of the Co-carbon bond is overcome to effect a trillion-fold acceleration in the homolysis rate (2) and how the high reactivity of radical intermediates are controlled to minimize side reactions.

Energetics of Interaction between the G-protein Chaperone, MeaB, and B12-dependent Methylmalonyl-CoA Mutase

“…2A) and had a specific activity of 54 mol succinyl-CoA formed/min/mg of protein at 37°C. This is ϳ40-fold higher than previously reported (7) and similar to the activity measured in our laboratory for the P. shermanii mutase (18,19).…”

Autoinhibition and Signaling by the Switch II Motif in the G-protein Chaperone of a Radical B12 Enzyme

“…Trifluoroacetic acid was purchased from Aldrich. Methylmalonyl-CoA was synthesized using malonylCoA synthetase and purified as described previously (38). The HPLC column used to quantify guanosine nucleotides was a Bondapak TM NH 2 10-m, 125 Å, 3.9 ϫ 300-mm column purchased from Waters.…”

A Human Vitamin B12 Trafficking Protein Uses Glutathione Transferase Activity for Processing Alkylcobalamins

“…The standards were prepared in the same buffer and treated analogously. The cobalamin products were analyzed as described (21). GSH and GSH-derived thioethers were analyzed as described (22).…”

“…The amino groups were derivatized with 2,4-dinitrofluorobenzene following reaction of free thiols with monoiodoacetic acid and injected onto a Bondapak TM NH 2 column (3.9 ϫ 300 mm, Waters) equilibrated with 4:1 (v/v) methanol/H 2 O. The column was eluted as described (21), and the eluant was monitored at 340 nm.…”