Stabilization of radical intermediates by an active-site tyrosine residue in methylmalonyl-CoA mutase

N H ThomäP F Leadlay


The adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the reversible rearrangement of methylmalonyl-CoA into succinyl-CoA by a free-radical mechanism. The recently solved X-ray crystal structure of methylmalonyl-CoA mutase from Propionibacterium shermanii has shown that tyrosine 89 is an active-site residue involved in substrate binding. The role of tyrosine 89, a conserved residue among methylmalonyl-CoA mutases, has been investigated by using site-directed mutagenesis to replace this residue with phenylalanine. The crystal structure of the Tyr89Phe mutant was determined to 2.2 A resolution and was found to be essentially superimposable on that of wild-type. Mutant and wild-type enzyme have very similar KM values, but kcat for the Tyr89Phe mutant is 580-fold lower than for wild-type. The rate of release of tritium from 5'-[3H]adenosylcobalamin during the enzymatic reaction and its rate of appearance in substrate and product were measured. The tritium released was found to partition unequally between methylmalonyl-CoA and succinyl-CoA, in a ratio of 40:60 when the reaction was initiated by addition of methylmalonyl-CoA and in a ratio of 10:90 when the reaction was initiated by addition of succinyl-CoA. The overall ...Continue Reading


Oct 5, 2001·Journal of Inorganic Biochemistry·Agnieszka Dybala-Defratyka, Piotr Paneth
Jul 29, 2006·Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences·R BanerjeePiotr Paneth
Apr 12, 2002·Chembiochem : a European Journal of Chemical Biology·S D WetmoreL Radom
May 25, 2010·Applied Microbiology and Biotechnology·Sun Mee HongTakahiro Kusakabe

Related Concepts

Methylmalonyl-CoA Mutase
Mutagenesis, Site-Directed
Plain X-ray
Free Radicals
Radicals (chemistry)
Crystallography, X-Ray
Intramolecular Transferase Activity

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