Protection of Radical Intermediates at the Active Site of Adenosylcobalamin-Dependent Methylmalonyl-CoA Mutase^,

Abstract

Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the interconversion of methylmalonyl-CoA and succinyl-CoA via radical intermediates generated by substrate-induced homolysis of the coenzyme carbon-cobalt bond. From the structure of methylmalonyl-CoA mutase it is evident that the deeply buried active site is completely shielded from solvent with only a few polar contacts made between the protein and the substrate. Site-directed mutants of amino acid His244, a residue close to the inferred site of radical chemistry, were engineered to investigate its role in catalysis. Two mutants, His244Ala and His244Gln, were characterized using kinetic and spectroscopic techniques. These results confirmed that His244 is not an essential residue. However, compared with that of the wild type, k(cat) was lowered by 10(2)- and 10(3)-fold for the His244Gln and His244Ala mutants, respectively, while the K(m) for succinyl-CoA was essentially unchanged in both cases. The primary kinetic tritium isotope effect (k(H)/k(T)) for the His244Gln mutant was 1.5 +/- 0.3, and tritium partitioning was now found to be dependent on the substrate used to initiate the reaction, indicating that the rearrangement of the substrate radical to the product radical was extremely slow. The His244Ala mutant underwent inactivation under aerobic conditions at a rate between 1 and 10% of the initial rate of turnover. The crystal structure of the His244Ala mutant, determined at 2.6 A resolution, indicated that the mutant enzyme is unaltered except for a cavity in the active site which is occupied by an ordered water molecule. Molecular oxygen reaching this cavity may lead directly to inactivation. These results indicate that His244 assists directly in the unusual carbon skeleton rearrangement and that alterations in this residue substantially lower the protection of reactive radical intermediates during catalysis.

Changes in the free energy profile of glutamate mutase imparted by the mutation of an active site arginine residue to lysine

“…In particular, we recently described the effects of mutating Arg-100, which forms a salt bridge with the γ-carboxylate of the substrate (Fig. 2) [28][29][30]. We compare the results of our present studies with the previous data, which affords some insights into the role of active site residues in promoting the rearrangement of substrate radicals in these enzymes.…”

“…For wild-type MMCM, tritium partitioning is independent of whether methylmalonyl-CoA or succinyl-CoA is the substrate, indicating that rearrangement of the substrate radicals is rapid compared with hydrogen transfer. However, two active site mutations, His244Gln and Tyr248Phe have been identified which cause tritium in AdoCbl to preferentially partition back to the substrate [29,30]. These mutations appear to exert effects very similarl to what we observe in the glutamate mutase Arg100Lys mutant.…”

“…The partitioning of hydrogen isotopes from an enzymebound intermediate between products and substrates provides an elegant way of investigating the relative heights of energetic barriers in an enzyme-catalyzed reaction. We have previously applied the same technique to investigate the partitioning of tritium at the 5'-carbon of AdoCbl between substrate and product in the wild-type enzyme [27]; tritium partition experiments have also been used to investigate the free energy profile of AdoCbl-dependent methylmalonyl-CoA mutase [28][29][30]. We compare the results of our present studies with the previous data, which affords some insights into the role of active site residues in promoting the rearrangement of substrate radicals in these enzymes.…”

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).…”

“…After the substrate enters the barrel, the barrel closes in around the substrate, implying that the active site should be inaccessible to other molecules (21). 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).…”