1e1c
From Proteopedia
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METHYLMALONYL-COA MUTASE H244A MUTANT
Overview
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.
About this Structure
1E1C is a Protein complex structure of sequences from Propionibacterium freudenreichii subsp. shermanii with B12 and DCA as ligands. Active as Methylmalonyl-CoA mutase, with EC number 5.4.99.2 Full crystallographic information is available from OCA.
Reference
Protection of radical intermediates at the active site of adenosylcobalamin-dependent methylmalonyl-CoA mutase., Thoma NH, Evans PR, Leadlay PF, Biochemistry. 2000 Aug 8;39(31):9213-21. PMID:10924114
Page seeded by OCA on Tue Nov 20 13:43:39 2007
