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

Abstract

Methylmalonyl-CoA mutase is an adenosylcobalamindependent enzyme that catalyzes the 1,2 rearrangement of methylmalonyl-CoA to succinyl-CoA. This reaction results in the interchange of a carbonyl-CoA group and a hydrogen atom on vicinal carbons. The crystal structure of the enzyme reveals the presence of an aromatic cluster of residues in the active site that includes His-244, Tyr-243, and Tyr-89 in the large subunit. Of these, His-244 is within hydrogen bonding distance to the carbonyl oxygen of the carbonyl-CoA moiety of the substrate. The location of these aromatic residues suggests a possible role for them in catalysis either in radical stabilization and/or by direct participation in one or more steps in the reaction. The mechanism by which the initially formed substrate radical isomerizes to the product radical during the rearrangement of methylmalonyl-CoA to succinyl-CoA is unknown. Ab initio molecular orbital theory calculations predict that partial proton transfer can contribute significantly to the lowering of the barrier for the rearrangement reaction. In this study, we report the kinetic characterization of the H244G mutant, which results in an acute sensitivity of the enzyme to oxygen, indicating the important role of this residue in radical stabilization. Mutation of His-244 leads to an ϳ300-fold lowering in the catalytic efficiency of the enzyme and loss of one of the two titratable pK a values that govern the activity of the wild type enzyme. These data suggest that protonation of His-244 increases the reaction rate in wild type enzyme and provides experimental support for ab initio molecular orbital theory calculations that predict rate enhancement of the rearrangement reaction by the interaction of the migrating group with a general acid. However, the magnitude of the rate enhancement is significantly lower than that predicted by the theoretical studies.

Novel Coenzyme B12-dependent Interconversion of Isovaleryl-CoA and Pivalyl-CoA

“…The steady formation of OH 2 Cbl during IcmF turnover under aerobic conditions suggests that inactivation might result from oxidative interception of cob(II)alamin (27). An alternative possibility is that inactivation is associated with loss of 5Ј-de- oxyadenosine from the active site, and the uncoupled cob(II)alamin is subsequently oxidized to OH 2 Cbl.…”

Nitric Oxide Inhibits Mammalian Methylmalonyl-CoA Mutase

“…Previous workers studying the Propionibacterium shermanii enzyme have shown that at ambient oxygen concentrations (ϳ200 M), oxygen reacts with the Cbl(II) generated during the enzymatic reaction, albeit at a slow rate (40). Other workers found no inhibition of the P. shermanii enzyme under ambient conditions, but enzyme activity was measured over only a 3-min interval (41). We found that at 200 M, oxygen had a small but statistically non-significant effect on the activity of mammalian methylmalonyl-CoA mutase during a 10-min incubation, but at 600 M oxygen significantly inhibited enzyme activity (Fig.…”

“…However, this may not apply to non-polar diatomic molecules like oxygen or NO, as oxygen has been shown to react with Cbl(II) generated by the P. shermanii enzyme, albeit at a slow rate (40). Mutation of the P. shermanii mutase at histidine 244 markedly increases the sensitivity of the enzyme to oxygen, indicating that this residue is critical for protecting the Cbl(II) and deoxyadenosyl radical intermediates (40,41). Modeling the possible structure of the homodimeric human enzyme against the known structure of the heterodimeric P. shermanii enzyme using the Swiss Model program suggests that the active sites of the two enzymes are similar, but such modeling can overlook subtle structural differences that could lead to increased sensitivity to NO and oxygen (52).…”

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

“…computational studies on methylmalonyl-CoA mutase have begun to provide insights into these issues (3)(4)(5)(6)(7)(8). They point to the critical role played by individual active site residues in labilizing the Co-carbon bond (7), and in shielding radical intermediates from unwanted side-reactions (4,6,8).…”

“…They point to the critical role played by individual active site residues in labilizing the Co-carbon bond (7), and in shielding radical intermediates from unwanted side-reactions (4,6,8).…”